WO2000065052A1

WO2000065052A1 - Bridge-1, a transcription factor

Info

Publication number: WO2000065052A1
Application number: PCT/US2000/010616
Authority: WO
Inventors: Joel F. Habener; Melissa K. Thomas; Gordon G. Wong; Kwok-Ming Yao
Original assignee: Habener Joel F; Thomas Melissa K; Wong Gordon G; Yao Kwok Ming
Priority date: 1999-04-22
Filing date: 2000-04-21
Publication date: 2000-11-02
Also published as: AU4649600A; WO2000065052A9

Abstract

The present invention concerns a transcription factor, Bridge-1. More specifically, isolated nucleic acid molecules are provided encoding Bridge-1. Recombinant methods for making Bridge-1 polypeptides are also provided as are Bridge-1 antibodies. Screening methods are also provided for identifying polypeptides that bind to Bridge-1 or a Bridge-1 fragment, such as a fragment containing PDZ-like binding domain of Bridge-1.

Description

BRIDGE-1 , A TRANSCRIPTION FACTOR

Statement as to Rights to Inventions Made Under Federally-Sponsored Research and Development

Part of the work performed during development of this invention utilized U.S. Government funds. The U.S. Government may have certain rights in this invention.

Cross-Reference to Related Application

This application claims the benefit of U.S. Provisional Application No. 60/130,490, filed April 22, 1999, the entire contents of which is hereby expressly incorporated herein by reference.

Field of the Invention

The present invention relates to the field of transcription factors. More specifically, the present invention relates to a novel mammalian transcription factor, to the nucleotide and amino acids sequences thereof, and to the use of this novel transcription factor in the diagnosis and treatment of developmental disorders including diabetes.

Background of the Invention

Basic helix-loop-helix (bHLH) transcription factors regulate a diverse array of physiologic processes in developing and adult organisms. Myogenesis, lymphocyte differentiation, neurogenesis, sex determination, and the development and functions of pancreatic β-cells are dependent on the regulated action of both ubiquitous and tissue-specific bHLH proteins (Murre, C., etal, Biochim. Biophys. Ada 1218:129-135 (1994), Naya, F. J., etal., Genes Development 77:2323-2334 ( 1997)). The ubiquitous E2 A family of proteins, including E 12 and E47, function either as homodimers or as heterodimers with tissue-specific Class B bHLH proteins to bind and transactivate promoters via conserved sequence elements known as E boxes. Although E2A proteins are best characterized in their interaction with other bHLH proteins, other types of protein-protein interactions have been described (Eckner, R. et al., Genes and Development 10:2418-2490 (1996); Kho, C.-J., et al., J. Biol. Chem. 272:3845-3851 (1997); Kho, C.-J., et al. J. Biol. Chem. 272:13426-13431 (1997); Loveys, D. A., etal, Gene 20TΛ69-

177 (1997); Qiu, Y., etα ., Mol. Cell. Biol. 75:2957-2964 (1998)). The activation of gene transcription by E2A may occur directly by the binding of bHLH dimers to DNA, via synergistic interactions with proteins bound to adjacent DNA, or by interaction with coactivating proteins. One experimental model system for the transactivation of transcription by

E2A is the pancreatic β-cell, in which the rat insulin I gene is regulated by glucose-responsive minienhancers consisting of E box binding sites for E2A (Nelson, C, et al., Genes Dev. 4:1035-1043 (1990)) and A boxes that bind homeoproteins (German, M. S. et al., Genes Dev. 6:2X65-2X16 (1992)). At the E boxes, E2A forms heterodimers with tissue-specific bHLH transcription factors such as Beta-2/NeuroD (Naya, F. J., et al, Genes Dev. 9: 1009-1019 (1995)). Homeoproteins, such as PDX-1, Lmxl, and Isl-1, bind to the A boxes and act in synergy with E2A heterodimers on adjacent E boxes to activate transcription of the insulin gene (German, M. S. etal., Genes Dev. 6:2X65-2X16 (1992); Johnson, J. D., et al., Mol. Cell. Biol. 7:3488-3496 (1997); Peers, B., et al., Mol. Endo.

5:1798-1806 (1994)).

The importance of these regulatory factors for both the development and function of β-cells is illustrated by several lines of investigation. Targeted disruption of beta-2/neuroD in mice results in abnormalities of islet development and diabetes mellitus (Naya, F. J., et al., Genes Development 1 1 :2323-2334 (1997)). Inactivation of E2A does not impair pancreatic development, but compensation by other members of the E2A family may be responsible for this observation (I tkin-Ansari, P., etal, Endocrinol. 737:3540-3543 (1996); Sharma, A., et al, Mol. Endocrinol. 77:1608-1617 (1997)). In both mice and humans, homozygous disruption of thepdx-1 gene arrests pancreatic development, whereas heterozygous disruption results in a diabetic phenotype (Dutta, S. et al, Nature 392:560 (1998); Jonsson, J. L. etal, Nature 371:606-609 (1994); Offield, M. F., et al., Development 122:983-995 (1996); Staffers, D. A., et al, Nature Genet. 77:138-139 (1997); Staffers, D. A., et al, Nature Genet. 75:106-110 (1997)). Additional factors also regulate the transactivational activities of E2 A in the pancreatic β-cell. Recent reports suggest that CBP and p300 may act as E2A coactivators (Eckner, R. et al, Genes and Development 70:2478-2490 (1996); Qiu, Y., et al, Mol. Cell. Biol 18:2951-2964 (1998)). p300 serves as a coactivator for both E2A and Beta-2 NeuroD in insulin gene transcription (Qiu, Y., et al, Mol. Cell. Biol. 18:2951-2964 (1998)).

PDZ domains, named for three proteins in which the motif was initially noted (post-synaptic density protein PSD-95 (Cho, K.-O., et al, Neuron 9:929- 942 (1992)), Drosophila discs-large tumor suppressor protein DlgA (Yun, C. H. C, etal, Proc. Natl. Acad. Sci. USA 94:3010-3015 (1997))), and the mammalian tight junction protein ZO-1 (Itoh, M., et al, J. Cell Biol. 727:491-502 (1993))), are conserved domains that mediate protein-protein interactions in a variety of intracellular signaling processes (Saras, J., and C.-H. Heldin, Trends in Biochem. Sci. 27:455-458 (1996)). For example, PDZ domains have been implicated in protein-protein interactions required for post-synaptic density ion channel and receptor clustering, signal transduction pathways regulating cell growth, visual signal transduction cascade regulation, and Fas-mediated regulation of apoptosis (Saras, J., and C.-H. Heldin, Trends in Biochem. Sci. 27:455-458 (1996)). PDZ domains appear in proteins with a diverse range of functions, including protein tyrosine phosphatases, proteases, ion channels, and signal transduction scaffolding molecules (Saras, J., and C.-H. Heldin, Trends in Biochem. Sci. 27:455-458 (1996)).

Proteins in the E2A family of basic helix-loop-helix transcription factors are important in a wide spectrum of physiologic processes as diverse as neurogenesis, myogenesis, lymphopoeisis, and sex determination. In the pancreatic β-cell, E2A proteins, in combination with tissue-specific transcription factors, regulate expression of the insulin gene and other genes critical for β-cell function.

Diabetes mellitus type I, or insulin-dependent diabetes, results from a genetically conferred vulnerability that causes a primary deficiency of insulin. This deficiency of insulin is believed to be the consequence of destruction of a specialized population of cells that produce insulin in the body, i.e., pancreatic β -cells. An autoimmune process may also contribute to β-cell damage. As a consequence of insulin lack (and glucagon excess), glucose production is augmented, and the efficiency of peripheral glucose use is reduced until a new equilibrium between these processes is reached at a very high plasma glucose level. Because of the high plasma glucose levels, the filtered load of glucose exceeds the renal tubular capacity for reabsorption. Glucose therefore is excreted in the urine in large quantities, causing, by its osmotic effect, increased excretion of water and salts and frequent urination. The goal of insulin treatment is to systematically lower plasma levels of glucose, free fatty acids and ketoacids to normal and reduce nitrogen losses. This result is achieved by direct actions of insulin and also by diminishing the secretion of the insulin antagonist glucagon.

Another more common form of diabetes mellitus, noninsulin-dependent or type II, often is associated with obesity. In this disease, there appears to be both a deficit in insulin production in combination with a resistance to the action of insulin on major target tissues. The locus of resistance is distal to the insulin receptor binding site, but defects in receptor tyrosine kinase activity, glucose transport, and activities of insulin-sensitive enzymes have been found. In addition, there is a derangement in β-cell recognition of glucose as a stimulus, so that first phase insulin secretion is lost, though a delayed release does occur. Treatment of type II diabetes does not normally require insulin administration. Caloric regulation, weight reduction if obesity is present, and the use of sulfony lurea drugs simultaneously improve tissue responsiveness to endogenous insulin and β-cell responsiveness to glucose. In late stages, insulin administration is usually required.

Increasing evidence from human genetic studies suggests that dysfunction of pancreatic beta cell transcriptional regulators may cause diabetes mellitus. Maturity onset diabetes of the young (MODY) is amonogenic heritable subgroup of type II diabetes with early onset (age 30 or less). Of the five identified genes with mutations linked to MODY in human families, four are transcription factors: HNF-lα, HNFl-β, HNF4α, and PDX-1 (Chevre, J.-C. et al., Diabetologia 47:1017-1023 (1998). These transcription factors function in both pancreatic development and in the differentiated pancreatic beta cell. The homeoprotein PDX- 1 is a key regulator of both pancreatic development and glucose-responsive transcription of the insulin gene. Mutations in PDX-1 may lead to diabetes in humans via disrupting the regulation of the insulin gene and/or altering the development of the pancreas and of pancreatic beta cell mass. Transcriptional regulators of the insulin gene and of pancreas development may be important targets for novel therapeutic agents of diabetes mellitus.

Insulin excess is usually caused by tumors of the β-cells. The cardinal manifestation is a low plasma glucose level in the fasting state. With chronic insulin excess and persistent hypoglycemia, disturbed central nervous system function results in bizarre behavior, defects in cerebration, loss of consciousness, or convulsions. Removal of the tumor may cure the condition. Alternatively, drugs that inhibit insulin secretion may ameliorate the condition.

There exists a need in the art for additional methods of treating all types of diabetes, preferably such methods would avoid the need for regular injections of insulin. Moreover, it would be advantageous if such methods were able to control endogenous insulin production by regulating insulin gene expression. Summary of the Invention

Cognizant of the need to identify new factors that might regulate insulin gene transcription, the inventors have discovered a novel transcription factor, Bridge-1. Bridge-1 is a novel coactivator for members of the E2A transcription factor family isolated from pancreatic insulinoma cells. Bridge-1 represents a novel PDZ-like domain coactivator for E2A and participates in the regulation of insulin gene transcription in pancreatic β-cells.

Bridge-1 interacts with E2A proteins to function as a coactivator of gene transcription mediated by E12 and E47. Rat Bridge-1 was isolated by yeast two- hybrid screening of a cDNA library prepared from rat insulinoma (INS- 1 ) cells, on the basis of its specific interaction with E12. Bridge-1 is homologous to proteins of unknown function from several species and contains a truncated PDZ-like domain, a domain known to be involved in protein-protein interactions. Bridge- 1 RNA is widely expressed in pancreatic islet cell lines and in a variety of murine and human tissues. By immunocytochemistry, the Bridge-1 protein expression pattern is primarily nuclear, with marked expression in pancreatic islets and coexpression with insulin. The interaction of Bridge-1 with E2A proteins is further demonstrated by coimmunoprecipitation of in vitro translated Bridge- 1 and El 2 and E47 and by mammalian two-hybrid studies. In contrast, Bridge-1 does not interact with the pancreas-specific basic helix-loop-helix protein Beta-

2/NeuroD. The PDZ-like domain of Bridge- 1 is required for interaction with E 12, as deletion mutants of Bridge- 1 that lack the PDZ-like domain interact poorly with El 2 in mammalian two-hybrid studies. The carboxy terminus of E12 participates in this interaction, as demonstrated by an impaired interaction with Bridge-1 of a truncated E 12 mutant (E 12ΔC) in which the carboxy-terminal 9 amino acids were deleted. Bridge-1 has transactivation potential, as a Gal4 DNA-binding domain/Bridge- 1 fusion protein transactivates a Gal4C AT reporter. Bridge-1 also functions as a coactivator by enhancing El 2- or E47-mediated activation of the rat insulin I gene minienhancer promoter-reporter constructs in transient transfection experiments. Substitution of the mutant El 2ΔC for El 2 reduces the coactivation of rat insulin I promoter-reporter constructs by Bridge-1. Inactivation of endogenous Bridge- 1 in insulinoma (INS- 1 ) cells by expression of a Bridge- 1 antisense RNA diminishes rat insulin I promoter activity . Bridge- 1 , by utilizing its PDZ-like domain to interact with E 12, provides a new mechanism for the coactivation and regulation of transcription of the insulin gene.

Bridge-1 signaling is not restricted to the fully developed pancreatic beta cell, but is also operative during pancreas development. Bridge-1 expression can be detected in the developing mouse pancreas at el 0.5 by rt-PCT and at el 5 by immunocytochemistry. Analysis of additional Bridge-1 mutants in transient transfection studies indicates that both the intact PDZ-like domain and the carboxyl terminal domain of Bridge-1 are required to mediate transcriptional transactivation. Bridge-1 also interacts with and appears to regulate other transcriptional activators. The pancreatic beta cell-specific transcription factor PDX- 1 interacts with Bridge- 1 in both yeast and mammalian two-hybrid systems.

In transient transfection experiments, Bridge- 1 transactivation activity is enhanced by addition of the coactivator p300, in a dose-dependent manner. The protein- protein interaction and transactivation functions of Bridge-1 may also be modulated by the multiple forms of Bridge-1 observed on Northern blots, in cDNA expression libraries, and on Western blots.

Thus, in one embodiment, the present invention is directed to isolated nucleic acid molecules comprising a polynucleotide encoding Bridge-1 whose amino acid sequence is shown in Figure 1 (SEQ ID NO:2), or a fragment thereof. In another aspect, the invention provides isolated nucleic acid molecules encoding Bridge- 1 having an amino acid sequence as encoded by the cDNA in the plasmid pcDN A3 -Bridge-1 deposited with the American Type Tissue Culture ("ATCC"), 10801 University Blvd. Manassas, V A, on April 20, 1999, and assigned accession number 203947.

In another embodiment, the invention is directed to an isolated nucleic acid molecule that hybridizes under stringent conditions to the above-described nucleic acid molecules. The present invention is also directed to variants of the nucleic acid molecules of the present invention, which encode fragments, analogs or derivatives of the Bridge-1 protein, e.g., polypeptides having at least one biological activity that is substantially similar to at least one biological activity of the Bridge-1 protein.

The present invention is further directed to isolated nucleic acid molecules that encode a Bridge-1 polypeptide as well as methods for generating nucleic acid molecules that encode a Bridge-1 polypeptide.

Further embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, and more preferably at least 95%, 96%, 97%, 98%, or 99% identical to the above described nucleic acid molecules.

The present invention also relates to vectors which contain the above- described isolated nucleic acid molecules, host cells transformed with the vectors and the production of Bridge-1 polypeptides by recombinant methods.

The present invention further provides isolated Bridge-1 polypeptides having the amino acid sequence shown in Figure 1 (SEQ ID NO:2). In a further aspect, isolated Bridge-1 polypeptides are provided having an amino acid sequence as encoded by the cDNA in the plasmid pcDN A3 -Bridge-1 deposited with the American Type Tissue Culture ("ATCC"), 10801 University Blvd. Manassas, NA, on April 20, 1999, and assigned accession number 203947.

In another embodiment, the invention is directed to screening methods for identifying proteins, proteins fragments, biological and chemical compounds and other small molecules that bind to the full-length Bridge-1 protein or Bridge-1 polypeptide fragments.

In another embodiment, the invention is directed to methods for identifying proteins, protein fragments, biological and chemical compounds, or other small molecules that enhance or inhibit Bridge-1 activity. In another embodiment, the invention is directed to a method for stimulating gene expression in a cell comprising transfecting said cell with a recombinant vector, said vector comprising an isolated polynucleotide having the nucleotide sequence set forth in FIG. 1 (SEQ ID NOT). In another embodiment, the invention is directed to a method for enhancing transcription activation of a Bridge-1 target gene. By "Bridge-1 target gene" is meant any gene transcription of which is mediated either directly or indirectly by Bridge-1, either as a transactivator, coactivator or otherwise.

In another embodiment, the invention is directed to a method for enhancing or inhibiting transcriptional activation of a Bridge-1 target gene by administering an effective amount of a protein, protein fragment, biological or chemical compound or other molecule that enhances or inhibits Bridge- 1 activity.

In another embodiment, the invention is directed to a method of treating diabetes comprising administering to a patient in need thereof a therapeutically effective amount of Bridge- 1 in a pharmaceutically acceptable carrier or excipient for a time sufficient to provide an effective level of endogenous insulin in the patient.

In another embodiment, the invention is directed to anti-Bridge- 1 antibodies.

Brief Description of the Figures

FIG. 1. Sequence of rat Bridge-1 cDNA and encoded protein. Figure 1 shows the sequence of rat Bridge-1 cDNA (SEQ ID NO: l) and the corresponding encoded protein (SEQ ID NO:2). Positions of in-frame stop codons are designated by asterisks under the corresponding nucleotide sequences. The arrowhead is placed below the corresponding nucleotide sequence to indicate the starting position of the two-hybrid clone # 18. The PDZ-like domain (i. e. , amino acids 138-178) is underlined. FIG. 2A. Homologies between Bridge-1 protein sequence and sequences from other species. Sequences used for alignment are the following: Bridge, rat Bridge-1 sequence (SEQ ID NO: 2); p27 human (SEQ ID NO:3), human proteasomal modulator subunit p27 (Genbank ace. no. AB003177); C. elegans (SEQ ID NO:4), sequence from chromosome III of C. elegans (Woods,

D. F., and P. J. Bryant, Ce/7 66Λ5X-464 (1991)) (Genbank ace. no. U23453); S. cerevisiae (SEQ ID NO:5), hypothetical 24.8 kD protein in FAA3-BET1 intergenic region from S. cerevisiae (Genbank ace. no. P40555). Amino acid similarities as determined by Blast analysis are shown as grey boxes and identities as bold print.

FIG. 2B. PDZ-like domain homologies between Bridge-1 and other PDZ-domain containing proteins. The Bridge-1 protein (SEQ ID NO: 2) is schematically depicted to illustrate the PDZ-like domain (PDZ) identified by homology with other proteins. Alignment positions with conserved similar or identical residues by Blast analysis in at least 50 percent of the aligned sequences are indicated with asterisks and grey boxes. Identical amino acids are designated in bold print. Protein sequences represented are the following: BRIDGE, rat Bridge-1, aa 138-178); SIP1 (SEQ ID NO: 6), interacting protein with human SRY (Poulat, F., etal, J. Biol. Chem. 272:1X61-1X12 (1997)) (Genbank ace. no. U82108), PDZ-domain A, aa 36-76, PDZ-domain B, aa 176-216; CLIM 1 , human carboxyl terminal LIM domain protein (Genbank ace. no. U90878), aa 30-65/76-80 (SEQ ID NO: 8); TAXINT, human Tax interaction protein 1 (Rousset, R., et al., Oncogene 16:643-654 (1998)) (Genbank ace no. AF028823), aa 49-89 (SEQ ID NO: 9); ZIP, human zipper containing protein (Dixon, B., B. etal, Biochim. Biophys. Ada 727(5:321-324 (1993)) (Genbank ace. no. 631508), aa 76-116 (SEQ ID NO:10); NHERF, rabbit protein cofactor that mediates protein kinase A regulation of the renal brush border membrane Na+/H+ exchanger (Wilson, R., et al, Nature 368:32-38 (1994); Zhuang, Y., et al, Cell 79:875-884 (1994)) (Genbank ace. no. U19815) PDZ-domain A, aa 39-79 (SEQ ID NO:l l), PDZ-domain B, aa 179-219 (SEQ ID NO: 12); ZO-1, mouse tight junction protein ZO-1 (Itoh, M., et al, J. Cell Biol. 727:491-502 (1993)) (Genbank ace. no. P39447), aa 447-487 (SEQ ID NO: 13); SERPROT, Aquifex aeolicus periplasmic serine protease (Deckert, G., et al, Nature 392:353-358 (1998)) (Genbank ace. no. AE000741), aa 280-318 (SEQ ID NO: 14); PROTHHO, Synechocystis sp. protease HhoB (Genbank ace. no. D9091 1), aa

346-384 (SEQ ID NO: 15); PROTDEGS, E. coli protease DEGS precursor (Genbank ace. no. P31137), aa 284-305 (SEQ ID NO: 16); and PDZK1, human PDZ domain containing-protein (Kocher, O., et al, Lab. Invest. 75: 1 17-125 (1998)) (Genbank ace. no. AF012281), aa 403-443 (SEQ ID NO: 17)). FIG. 2C. Comparison of the Bridge-1 PDZ-like domain with typical

PDZ domain sequences. Alignment of typical PDZ domains and designation of regions of secondary structure (boxed) are depicted according to Doyle et al. (Doyle, D. A., et al, Cell 55: 1067-1076 (1996)). A segment of Bridge-1 sequence is aligned for comparison. Amino acids within Bridge-1 sequence identified as conserved among sequences aligned in FIG.2b are designated (*) for reference. For each aligned position, similarities or identities by Blast analysis between the Bridge-1 sequence and any of the three ZO-1, PSD95-3, or DLG-1 sequences are indicated (+). Amino acid sequences illustrated are as follows: Bridge-1, aa 105-190; ZO-1, mouse tight junction protein ZO-1 (Itoh, M., et al, J. Cell Biol. 727:491-502 (1993)) (Genbank ace. no. P39447), aa 423-501 (SEQ

ID NO: 18); PSD95-3, rat presynaptic density protein 95, PDZ domain 3 (Cho, K.-O., et al, Neuron 9:929-942 (1992)) (Genbank ace. no. P31016), aa 312-391 (SEQ ID NO: 19); DLG-1, D. melanogaster lethal (Anand, G., X. et al, J. Biol. Chem. 272:19140-19151 (1997)) discs large- 1 tumor suppressor protein (Yun, C. H. C, etal, Proc. Natl. Acad. Sci. USA 94:3010-3015 (1997)) (Genbank ace. no.

P31007), aa 485-564 (SEQ ID NO: 20).

FIG. 3 (Panels A-D). Characterization of Bridge-1 Expression. A) Northern blot of Bridge- 1 transcript in total RNA from rodent cell lines (upper panel). For each lane, 20 ug of total RNA derived from the following cell lines was hybridized with ³²P-labeled Bridge-1 cDNA : INS- 1, rat insulinoma cells; RIN 1027-B2, rat islet tumor somatostatin-secreting cells; RIN 1046-38, rat insulinoma cells; RIN 56A, rat insulinoma cells; HepG2, human hepatoblastoma cells; PC 12, rat pheochromocytoma cells; AR42J, rat exocrine pancreatic tumor cells; InRl-G9, hamster islet tumor glucagon-secreting cells; HIT-T15, hamster insulinoma cells; αTC 1 , mouse islet tumor glucagon-secreting cells; βTC6, mouse islet tumor insulin-secreting cells. The blot was stripped and reprobed with a γ-actin cDNA as a loading control (lower panel).

B) Northern blot of Bridge-1 transcript in mouse tissues. A commercially generated Northern blot (Clontech) containing 2 μg of poly (A)+ RNA per lane from the murine tissues indicated was hybridized with ³²P-labeled

Bridge-1 cDNA.

C) Northern blot of Bridge-1 transcript in human endocrine tissues. A commercially generated Northern blot (Clontech) containing 2 μg of poly (A)+ RNA per lane from the human tissues indicated was hybridized with ³²P-labeled Bridge-1 cDNA.

D) Autoradiogram after SDS-PAGE fractionation of ³⁵S-labeled in vitro translated Bridge-1 protein (left panel). Western blot analysis of in vitro translated Bridge-1 after SDS-PAGE fractionation (right panel).

FIG. 4 (panels A-D). Immunocytochemical staining of Bridge-1 in RIN1027-B2 cells and mouse pancreas. Fluorescent immunostaining of

RIN 1027-B2 cells was conducted with rabbit polyclonal anti-Bridge- 1 antisera (A) or preimmune antisera (C) and photographed under identical conditions. A phase contrast view of the field of cells stained with anti-Bridge- 1 antisera (B) is shown for comparison. Mouse embryonic day 19 pancreas (D) was stained with rabbit polyclonal anti-Bridge- 1 antisera. An islet (*), ducts (d), and adjacent exocrine pancreas are shown. Examples of exocrine cell nuclei with positive Bridge-1 immunostaining are indicated with arrows.

FIG. 5 (Panels A-D). Bridge-1 is co-expressed with insulin in murine pancreas. Fluorescent immunostaining of adult murine pancreas was conducted by co-staining with rabbit polyclonal anti-Bridge- 1 antiserum (A) and guinea pig anti-insulin antiserum (B). Co-staining of an adjacent section of murine pancreas with rabbit preimmune antiserum (C) and guinea pig anti-insulin antiserum (D), photographed under identical conditions, is shown for comparison. Arrows point to β cells that coexpress Bridge-1 and insulin within an islet of Langerhans. FIG. 6 (Panels A-B). Coimmunoprecipitation of Bridge-1 and E2A proteins.

A) ³⁵S-labeled in vitro translated E 12 was incubated with cold in vitro translated Bridge-1 prior to immunoprecipitation with anti-Bridge- 1 rabbit polyclonal antisera (upper panel) or preimmune antisera (lower panel). B) Immunoprecipitation reactions with anti-Bridge- 1 antisera were conducted with ³⁵S-labeled in vitro translated E12 or E47 in the presence (upper panel) or absence (lower panel) of cold in vitro translated Bridge-1. Autoradiograms of the immunoprecipitation reactions after SDS-PAGE fractionation are shown. In vitro translated E 12 and E47 migrated on SDS-PAGE at approximately 69 kD and 68 kd, respectively.

FIG. 7. Bridge-1 interacts with El 2 in a mammalian two-hybrid system. HeLa cells were transiently transfected with 5 μg Gal4CAT reporter and 10 μg of pM (Gal4DBD), pM-Bridge (Gal4DBD-Bridge-l), pNP16 (NP16AD) or PNP16-E12 (NP16AD-E12), as designated. Results shown are the mean ± SEM of 5 transfections (n=5), conducted in duplicate.

FIG. 8. Bridge-1 and Beta-2/NeuroD do not interact in a mammalian two-hybrid system. HeLa cells were transiently transfected with 5 μg Gal4CAT reporter and 10 μg of pM (Gal4DBD). pM-Beta-2 (Gal4DBD-Beta-2), pNP16 (NP 16AD), pNP 16-E 12 (VP 16 AD-E 12), or pNP 16-Bridge- 1 (NP 16AD-Bridge), as designated. Results shown are the mean ± SEM of 2 transfections conducted in duplicate.

FIG. 9. The Bridge-1/E12 interaction requires the PDZ-like domain of Bridge-1. HeLa cells were transiently transfected with Gal4CAT reporter, pM, pM-Bridge-1, pNP16 (NP16AD) or pNP16-E12 (NP16AD-E12), as in FIG. 7. pM-Bridge-l(l-72), pM-Bridge-l(l-133), and pM-Bridge-l(l-184) were substituted for pM-Bridge-1. The interaction of each mutant with pNPl 6-E12 was assessed and normalized to the interaction seen for the full length pM-Bridge-1 and PNP16-E12. Aliquots of the transfected cell extracts were assessed by Western blotting to confirm comparable expression of the pM-Bridge-1 constructs tested. Results shown are the mean ± SEM of 4 transfections (n=4), conducted in duplicate.

FIG. 10 (Panels A-B). The carboxy-terminus of El 2 contributes to the Bridge-1/E12 interaction.

A) Schematic diagrams of E 12 (aa 1 -649), the E 12 fragment utilized as bait in the yeast two-hybrid screening that identified Bridge- 1 (aa 521 -649), and the E12 mutant E12ΔC (aa 1-640). The two activation domains are designated as AD 1 and AD2 and the basic helix-loop-helix domain as bHLH (modeled after a published schematic diagram ) (Spicer, D. B., et al, Twist. Science 272:1416- 1480 (1996)). The carboxy terminal sequences of E12, E12 bait, and E12ΔC are shown below the respective schematic diagrams. The asterisk designates the carboxy terminus of E12ΔC, truncated by nine amino acids (EAHΝPAGHL), due to the introduction of a TAG stop codon corresponding to amino acid position 641 in E12.

B) HeLa cells were transiently transfected with Gal4CAT reporter, pM, pM-Bridge- 1 , pNP 16 (NP 16 AD) or pNP 16-E 12 (NP 16 AD-E 12), as in FIG.

7. pNPl 6-E 12ΔC was substituted for pNP16-E12 and assessed for interaction with pM-Bridge- 1. The observed interaction was normalized to that seen for pNPl 6-E 12. Aliquots of the transfected cell extracts were assessed by Western blotting to confirm comparable expression of pNP16-E12 and pNP16-E12ΔC. Results shown are the mean ± SEM of 5 transfections (n=5), conducted in duplicate.

FIG. 11. Bridge-1 has intrinsic transactivation potential. BHK cells were transiently transfected with 1 μg Gal4CAT reporter and 4 μg pM (GaWDBD) or 4 μg pM-Bridge-1 (Gal4DBD-Bridge), as designated. Results shown are the mean ± SEM of 3 transfections (n=3) conducted in triplicate. FIG. 12 (panels A-D). Bridge-1 co-activation of rat insulin I minienhancer FarFlat reporter with E47 or E12.

A) Bridge-1 coactivation of FarFlat with E47. HeLa cells were transiently transfected with 2 μg 5FF1CAT reporter and 2 μg pcDNA3-E47, and/or 2 μg pcDNA3-Bridge-l, as indicated. pcDNA3 was added to normalize the amount of pcDNA3 vectors across all transfections. pBluescript was included to provide a total of 25 μg DNA per transfection. Results shown are the mean ± SEM of 6 transfections conducted in triplicate (n=5) or duplicate (n=l).

B) Dose-dependent Bridge-1 coactivation ofFarFlat with El 2. HeLa cells were transiently transfected with 2 μg 5FF 1 CAT reporter in the presence or absence of 1 μg pcDNA3-E12 and/or 1 to 4 μg pcDNA3-Bridge-l, as indicated. pcDNA3 was added to normalize the amount of pcDNA3 vectors across all transfections. pBluescript was included to provide a total of 25 μg DNA per transfection. Results shown are the mean ± SEM of triplicate samples. C) The El 2ΔC mutant diminishes Bridge-1 coactivation ofFarFlat.

HeLa cells were transiently transfected with 2 μg 5FF1CAT reporter in the presence of4 μg pcDN A3 -Bridge-1 and 1 μg pcDNA3-E12 orpcDNA3-E12ΔC. pBluescript was included to provide a total of 25 μg DNA per transfection. Results shown are the mean ± SEM of four transfections (n=4) conducted in duplicate and normalized to the activity of pcDN A3 -Bridge- 1 with pcDN A3 -E 12.

D) Representative Western blot demonstrating comparable expression of pcDN A3 -E 12 and pcDNA3-E 12ΔC . Aliquots of extracts from cells transfected in a representative experiment as described in FIG. 12C were subjected to

SDS-polyacrylamide electrophoresis followed by Western blotting with rabbit polyclonal antisera directed against El 2.

FIG. 13. Bridge-1 is expressed in developing mouse pancreas. Polyclonal rabbit anti-Bridge- 1 antisera was used in immunostaining of paraffin sections of mouse embryonic day 15 pancreas. At embryonic day 15, Bridge-1 is expressed in a nuclear pattern throughout the branching, developing ductal tree destined to become pancreas, as well as within structures budding from the ducts that likely represent developing islets. The arrowhead indicates nuclear Bridge-1 staining in one of the buds of the developing pancreatic ductal tree. FIG. 14 (Panels A and B). Bridge-1 interacts with PDX-1. (A) Bridge-1 interacts with PDX-1 in a yeast two-hybrid system. El 2 interacting clones identified by yeast two-hybrid screening were tested for their interaction with human interleukin-1 receptor (IL-R), D. melanogaster bicoid, PDX-1, and rat E 12 fusion proteins by semi-quantitative yeast two-hybrid interaction assay. Strength of interaction was measured by growth on leucine dropout plates to follow LEU reporter gene activity and by the intensity of blue color on X-gal plates to assess lacZ reporter gene activity, after 30 °C incubation for 72 hours.

A "-" denotes no growth on leucine dropout plates and white colonies on X-gal plates. A "++" indicates growth on leucine dropout plates and intense blue colonies on X-gal plates. (B) Bridge-1 interacts with PDX-1 in a mammalian two- hybrid system. HeLa cells were transiently transfected with 5 μg Gal4CAT reporter and 10 μg of pM (GaWDBD), pM-Bridge (GaWDBD-Bridge- 1 ), pNP 16

(NP16AD) or pNP16-PDX-l (NP16AD-PDX-1), as designated. When both Bridge-1 and PDX-1 fusion proteins were present, CAT activity was enhanced relative to the controls, consistent with a Bridge-1 /PDX-1 interaction.

FIG. 15. The PDZ-like domain is required for Bridge-1 transactivation activity. Proline residues were substituted for highly conserved amino acids in a series of point mutations constructed within the PDZ-like domain of the Bridge- 1 /GaW DΝA-binding domain fusion protein construct. BHK cells were transiently transfected, in duplicate, with 1 μg GaWCAT reporter and 4 μg pM (EMPTY VECTOR) or 4 μg pM-Bridge-1 (BRIDGE-1) or its mutants (N159P, N164P, N175P, D165P, G151P), as designated. A fluorimager scan of thin layer chromatography of a representative fluorimetric chloramphenicol acetyltransferase activity assay is shown. Asterisks (*) represent acetylated forms of the fluorescent substrate that results from chloramphenicol acetyltransferase activity. Quantitation of these transactivation activities relative to empty vector activity is as follows: Bridge-1, 854%; VI 59P, 96%; N164P, 335%; N175P, 122%; D156P, 44%; G151P, 185%. Note that each of the five Bridge-1 point mutants tested have diminished transactivation activity relative to the wild-type Bridge-1.

FIG. 16. p300 coactivates Bridge-1 transactivation activity. BHK cells were transiently transfected, in duplicate, with 1 μg GaWCAT reporter, 0.5 μg of empty vector pM or 0.5 μg pM-Bridge-1 , and 0.0 to 3.0 μg p300 expression plasmid, as indicated. Results shown are the mean of two transfections. p300 increases the Bridge- 1/Gal4 DNA-binding domain fusion protein transactivation of the GaWCAT reporter construct in a dose dependent manner.

FIG. 17. A "small form" of Bridge-1 is found on Western blots. Western blot analysis, utilizing rabbit polyclonal anti-Bridge- 1 antisera, of whole cell extracts derived from HeLa, BHK, and mouse insulinoma MIN-6 cells following SDS-PAGE fractionation and electroblotting onto a PNDF membrane. Enhanced chemiluminescence was utilized to visualize the protein antisera complexes. Migration positions for the full length Bridge- 1 (29kD) and the " small form" of Bridge-1 (19kD) are indicated. Note that the proportion of full length to "small form" Bridge-1 varies among the different cell types analyzed.

FIG. 18. The Bridge-1/E12 interaction requires the PDZ-like domain of Bridge-1. In a mammalian two-hybrid system, HeLa cells were transiently transfected with GaWDNA-binding domain Bridge- 1 or GaWDNA-binding domain/Bridge 1 deletion mutant (amino acids as indicated) fusion protein and

E12/NP16 activation domain fusion protein expression plasmids and a GaWCAT reporter. The interaction of each mutant with the E12/NP16 activation domain fusion protein was assessed and normalized to the interaction observed with the full length GaWDNA-binding domain/Bridge- 1 fusion protein. Data shown are the mean ± SEM of four transfections, conducted in duplicate. Bridge- 1 mutants that lacked an intact PDZ domain interacted poorly with El 2 while mutants with an intact PDZ domain retained the ability to interact with El 2.

FIG. 19. The Bridge-1/E12 interaction requires the PDZ-like domain of Bridge-1. In a yeast two-hybrid interaction system, Bridge-1 mutant fusion proteins (amino acids as indicated) were tested for the strength of their interaction with a rat El 2 fragment (amino acids 521-649) fusion proteins by measurement of β-galactosidase levels extracted from transformants. Results shown are the mean ± SEM of three independent determinations, conducted in duplicate. As in the mammalian two-hybrid system, Bridge-1 mutants that lacked an intact PDZ domain interacted poorly with E12 while mutants with an intact PDZ domain retained the ability to interact with El 2.

FIG. 20. The Bridge-1 PDZ domain is required for interaction with El 2. HeLa cells were transiently transfected with expression plasmids encoding fusion proteins of GaWDNA-binding domain (GaW), GaWDNA-binding domain/Bridge- 1 (Bridge) or GaWDNA-binding domain/Bridge- 1 proline substitution mutants (amino acid positions and changes as indicated) in conjunction with an expression plasmid encoding an E 12NP 16 activation domain fusion protein and a GaWCAT reporter. A representative fluorometric CAT assay, conducted in duplicate, is shown. Asterisks indicate the CAT activity as demonstrated by acetylated forms of the fluorescent substrate. Each of the five proline substitution mutations in conserved amino acids within the Bridge- 1 PDZ domain disrupts the ability of Bridge-1 to interact with E12 in this mammalian two-hybrid assay. These data indicate that these structural features of the Bridge- 1 PDZ domain are required for interaction with El 2. FIG. 21. Schematic diagram of El 2 mutant constructs tested in mammalian two-hybrid experiments for interaction with Bridge-1. The two activation domains are designated as AD1 and AD2 and the DΝA-binding and dimerization bHLH domain is designated bHLH. The carboxy-terminal sequences of E12, E12 bait, and E12ΔC are shown belowthe respective schematic diagrams. The asterisk designates the carboxy terminus of E12ΔC, truncated by 9 amino acids (EAHΝPAGHL), due to the introduction of a TAG stop codon by site- directed mutagenesis corresponding to amino acid position 641 in El 2.

FIG.22. Carboxy-terminal amino acids ofE12 mediate interaction with Bridge-1. HeLa cells were transiently transfected with GaWDNA-binding domain/Bridge- 1 fusion protein and E12NP16 activation domain or El 2 mutant/NP16 activation domain fusion protein expression plasmids and a GaWCAT reporter. The observed interactions in the mammalian two-hybrid assay were normalized to that seen for GaWDNA-binding domain/Bridge- 1 and E 12/NP 16 activation domain fusion proteins. Results shown are the mean ± SEM of three to five transfections conducted in duplicate. Deletion of 9 carboxy- terminal amino acids in E12 reduced its interaction with Bridge-1 by over 50 percent, and a larger deletion of E12 that included the bHLH domain eliminated the remaining interaction with Bridge-1.

FIG. 23. The E12ΔC mutant diminishes Bridge-1 coactivation of the rat insulin I promoter minienhancer FarFlat. HeLa cells were transiently transfected with a rat insulin I promoter-reporter construct encoding pentamerized FarFlat enhancer sequences and a CAT reporter and expression plasmids encoding Bridge- 1 , E 12 or the E 12ΔC mutant in which the carboxy-terminal 9 amino acids were deleted by mutagenesis. Data shown are the mean ± SEM of four transfections conducted in duplicate and normalized to the activity of Bridge-1 combined with E 12. The E 12ΔC mutation that impairs Bridge- 1 -E 12 interaction (see FIG. 22) decreases, by 65%, the combined activity of Bridge-1 and E12 on insulin promoter enhancer FarFlat. These data indicate that the strength of the protein-protein interaction modulates the observed coactivation. FIG. 24. Inactivation of Bridge-1 in insulin-producing cells reduces insulin promoter activity. Rat insulinoma (IΝS-1) cells were transiently transfected with an antisense Bridge-1 cDNA expression construct (antisense- Bridge-1) or the empty expression plasmid pcDNA3 (empty vector) and a rat insulin I promoter-reporter construct spanning residues -410 to +47 of the rat insulin I promoter sequence, including the minienhancer FarFlat. Expression of the Bridge-1 antisense construct decreased insulin promoter activity by 45%), indicating that endogenous Bridge- 1 contributes to insulin promoter activation in insulin-producing cells.

FIG. 25. Bridge-1 interacts with PDX-1. Recombinant glutathione-S- transferase/Bridge-1 fusion protein (GST-Bridge) or glutathione-S-transferase (GST) were incubated with [³⁵S]-radiolabeled in vitro translated rat PDX-1 protein and Glutathione Sepharose 4B (Pharmacia) beads. After multiple washes, proteins adherent to the glutathione sepharose beads were subjected to SDS- polyacrylamide electrophoresis. The corresponding autoradiogram demonstrating that GST-Bridge-I interacted with radiolabeled PDX-1 is shown.

FIG. 26. Bridge-1 activates the insulin promoter enhancer FarFlat in conjunction with PDX-1 and E2A proteins. HeLa cells were transiently transfected with expression plasmids for rat PDX- 1 , E 12, and E47 and a rat insulin I promoter-reporter construct encoding pentamerized FarFlat enhancer sequences and a CAT reporter. Transfections were conducted in the presence (solid bar) or absence (open bar) of an expression plasmid for Bridge-1. Data shown are the mean ± SEM of 6 transfections (n=6), each conducted in duplicate, and normalized to the CAT activity observed in the absence of Bridge- 1. The addition of Bridge-1 increased the activation of the insulin promoter enhancers by approximately 30 percent.

FIG. 27. Bridge-1 interacts with multiple domains within the nuclear receptor coactivator p300. Recombinant glutathione-S-transferase fusion proteins including fragments of human p300 (GST-p300, amino acids as indicated) were incubated with [ ⁵S]-radiolabeled in vitro translated rat Bridge-1 protein and Glutathione Sepharose 4B (Pharmacia) beads. After multiple washes, proteins adherent to the glutathione sepharose beads were subjected to SDS- polyacrylamide electrophoresis. The corresponding autoradiogram demonstrates that Bridge- 1 interacts with two distinct segments of p300, amino acids 1 -595 and 741-1571, but not with a third segment, amino acids 1572-2370. FIG 28. Bridge-1 interacts with multiple domains within the nuclear receptor corepressor NcoR. Recombinant glutathione-S-transferase fusion proteins including the two nuclear receptor binding domains of the nuclear receptor corepressor NCoR (GST-NCoR, NBDl or NBD2) were incubated with [³⁵S]-radiolabeled in vitro translated rat Bridge-1 protein and Glutathione Sepharose 4B (Pharmacia) beads. After multiple washes, proteins adherent to the glutathione sepharose beads were subjected to SDS-polyacrylamide electrophoresis. The corresponding autoradiogram demonstrates that Bridge-1 interacts with both nuclear receptor binding domains of NCoR.

FIG. 29. Bridge-1 conform multimers. Recombinant glutathione-S- transferase/Bridge-1 fusion protein (GST-Bridge) or glutathione-S-transferase

(GST) were incubated with [³⁵S]-radiolabeled in vitro translated rat Bridge-1 protein and Glutathione Sepharose 4B (Pharmacia) beads. After multiple washes, proteins adherent to the glutathione sepharose beads were subjected to SDS- polyacrylamide electrophoresis. The corresponding autoradiogram, demonstrating that GST-Bridge- 1 interacted with radiolabeled Bridge-1, is shown. The likely significance of a Bridge- 1 /Bridge-1 interaction is to provide a larger surface for docking of other interacting proteins to stabilize protein complexes in the regulation of transcription.

FIG. 30. The carboxy-terminal domain within Bridge-1 is required for transactivation. We analyzed several GaW DN A-binding domain/Bridge- 1 mutant fusion constructs for their ability to activate a GaWCAT reporter construct in transient transfections of BHK cells. Deletions of the carboxy terminus of Bridge- 1 markedly diminished the observed transactivation. For example, truncation of Bridge- 1 at amino acid 184 by site-directed mutagenesis with insertion of a premature stop codon eliminated Bridge- 1 transactivation (lower panel). This loss of transactivation was not due to decreased Bridge-1 fusion protein expression, as Western blotting of the transfected cell extracts with antiserum directed against the GaW DNA-binding domain demonstrated substantial expression levels of the mutant fusion protein (data not shown). This Bridge-1 (1-184) mutant was of interest, because it retained the PDZ homology domain and the ability to interact with E 12 in the mammalian two-hybrid studies in HeLa cells (upper panel) but was defective in transactivation activity (lower panel). These findings indicate that the carboxy terminus of Bridge-1 is required for transactivation and that the PDZ homology domain may function independently of the carboxy-terminal domain of Bridge-1 under certain experimental conditions. The PDZ domain may also participate in Bridge-1 transactivation because, in an independent series of experiments, proline-substitution mutagenesis of conserved amino acids within the PDZ domain substantially reduced Bridge-1 transactivation activity, indicating that the PDZ domain within Bridge-1 is also required for its transactivation activity.

Detailed Description of the Preferred Embodiments

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a novel mammalian transcription factor (Bridge-1) whose amino acid sequence is shown in Figure 1 (SEQ ID NO:2). The nucleotide sequence shown in Figure 1 (SEQ ID NO: 1 ) was obtained by sequencing a cDNA clone, which was deposited with the American Type Tissue Culture ("ATCC") and assigned accession number 203947.

Nucleic Acid Molecules

As used herein, a Bridge-1 "polynucleotide" refers to a molecule having a nucleic acid sequence contained in SEQ ID NOT . For example, the Bridge-1 polynucleotide can contain the nucleotide sequence of the full-length Bridge-1 cDNA sequence shown in FIG. 1 (SEQ ID NOT), including the 5' and 3 ' untranslated sequences, the coding region, with or without any signal sequence, the protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a Bridge-1 "polypeptide" refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.

In one embodiment of the present invention, isolated polynucleotides are provided which encode the Bridge- 1 protein. Using information provided herein, such as the nucleotide sequence in Figure 1 (SEQ ID NOT) or the above- described deposited clone, a nucleic acid molecule of the present invention encoding a Bridge- 1 polypeptide may be obtained using standard cloning and screening procedures. Illustrative of the invention, the nucleic acid molecule described in Figure 1 (SEQ ID NOT) was obtained from a cDNA expression library from rat pancreatic islet cells. The Bridge- 1 cDNA of the present invention encodes a protein of about 222 amino acids, which includes a PDZ-like domain.

Bridge-1 is widely expressed, since the corresponding transcript was found in several human tissues, including pancreas, testes, kidney, and liver.

Isolated nucleic acids of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.

By "isolated" nucleic acidmolecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment (e.g., the natural environment if it is naturally occurring), and thus is altered "by the hand of man" from its natural state. For example, recombinant DNA molecules contained in a vector are considered isolated for purposes of the present invention. Additional illustrative examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells and purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vitro RNA transcripts of the DNA molecules of the present invention as well as partially or substantially purified mRNA molecules. "Purified" as it refers to preparations made from biological cells or hosts should be understood to mean any cell extract containing the indicated DNA or protein including a crude extract of the DNA or protein of interest. For example, in the case of a protein, a purified preparation can be obtained by following an individual technique or a series of preparative or biochemical techniques and the DNA or protein of interest can be present at various degrees of purity in these preparations. The procedures may include for example, but are not limited to, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography, density gradient centrifugation and electrophoresis. "Substantially pure" should be understood to mean a "highly" purified preparation that contains at least 95% of the DNA or protein of interest. Isolated nucleic acid molecules according to the present invention further include nucleic acid molecules produced synthetically.

Isolated polynucleotides of the present invention include DNA molecules comprising an open reading frame (ORF), i.e. coding region, with an initiation codon at position 495 of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1); and DNA molecules which comprise a sequence substantially different than that described above but which, due to the degeneracy of the genetic code, still encode the Bridge-1 protein. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate the degenerate variants described above.

In another aspect, the invention provides isolated nucleic acid molecules encoding the Bridge- 1 polypeptide having an amino acid sequence as encoded by the cDNA clone in the plasmid pcDN A3 -Bridge-1 deposited with the American

Type Tissue Culture ("ATCC") and assigned accession number 203947. The invention further provides an isolated polynucleotide having the nucleotide sequence of the Bridge-1 coding region shown in Figure 1 (SEQ ID NO: 1 ) or the nucleotide sequence of the Bridge-1 cDNA contained in the above-described clone, or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated nucleic acid molecules, preferably DNA molecules, are useful as probes for gene mapping by in situ hybridization with chromosomes and for detecting expression of the Bridge-1 gene, or genes homologous to the Bridge- 1 gene, in human tissue, for instance, by Northern blot analysis.

In another aspect, the invention provides an isolated nucleic acid molecule that hybridizes under stringent conditions to the above-described nucleic acid molecules. As used herein "stringent conditions" is intended to mean, as a non- limiting example, overnight incubation at 42 °C in a solution comprising 50% formamide, 5xSSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lxSSC at about 65°C. Preferably, such "an isolated nucleic acid molecule that hybridizes under stringent conditions" will be at least 15 bp, preferably at least 20 bp, more preferably at least 30 bp, more preferably at least 40 bp, and most preferably, at least 50 bp in length.

As used herein, "fragments" of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA clone described above or the nucleotide sequence as shown in Figure 1 (SEQ ID NOT) or the nucleotide sequence of the ORF, i.e. the coding region, as shown in Figure 1 (SEQ ID NO: 1), is intended to mean DNA fragments at least 15 bp, more preferably at least 20 bp, more preferably at least 30 bp, more preferably at least 40 bp, more preferably at least 50 bp, more preferably at least 60 bp, more preferably at least 70 bp, more preferably at least 80 bp, more preferably at least 90 bp, more preferably at least 100 bp length, and most preferably at least 200 bp, in length. Such fragments are useful, wter alia, as diagnostic probes and primers. Larger DNA fragments, up to, for example, 500 bp in length, are also useful as probes according to the present invention. A fragment of at least 20 bp in length, for example, is intended to mean fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence of the ORF, i. e. coding region, as shown in FIG. 1 (SEQ ID NO: 1 ). As indicated, such fragments are useful diagnostically wter alia as a probe according to conventional DNA hybridization techniques or as primers for amplification of a target sequence by the polymerase chain reaction (PCR).

In a preferred embodiment, polynucleotide fragments of the invention comprise at least 15 contiguous nucleotides of the Bridge- 1 coding sequence shown in Fig. 1 (SEQ ID NOT), but do not comprise all or a portion of any Bridge-1 intron. In another embodiment, the nucleic acid comprising Bridge-1 coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5' or 3 ' to the Bridge-1 gene in the genome.)

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. The EST sequences referred to below were identified in a BLAST search of the EST database. These sequences are believed to be partial sequences of the cDNA insert identified in the recited GenBank accession numbers.

For example, the following sequences are related to the coding region of SEQ ID NO: 1, GenBank Accession Nos: AW140997 (SEQ ID NO:29); AI410372 (SEQ ID NO:30); AI710949 (SEQ ID NO.31); AI410370 (SEQ ID

NO: 32); AI175576 (SEQ ID NO:33); AI410377 (SEQ ID NO:34); AI176737 (SEQ ID NO:35); AI059501 (SEQ ID NO:36); AI577670 (SEQ ID NO:37); AI030624 (SEQ ID NO:38); W97405 (SEQ ID NO:39); W59260 (SEQ ID NO.40); AA990371 (SEQ ID NO.41); AV085226 (SEQ ID NO:42); AA458312 (SEQ ID NO:43); AA244824 (SEQ ID NO:44); W41287 (SEQ ID NO:45);

AA764187 (SEQ ID NO:46); AA530067 (SEQ IDNO:47); AA760338 (SEQ ID NO:48); AV239440 (SEQ ID NO:49); AA230657 (SEQ ID NO:50); AA832760 (SEQ ID NO.51); AN018936 (SEQ ID ΝO:52); AA0331 11 (SEQ ID NO:53); W41542 (SEQ ID NO:54); AI426803 (SEQ ID NO:55); AVI 17212 (SEQ ID NO:56); AI853315 (SEQ ID NO:57); W61442 (SEQ ID NO:58); AV043811

(SEQ ID NO:59); AI194159 (SEQ ID NO:60); AA958415 (SEQ ID NO:61); W77431 (SEQ ID NO:62); AA940225 (SEQ ID NO:63); AW495918 (SEQ ID NO:64); AV019366 (SEQ IDNO:65); AW124782 (SEQ IDNO:66); AW496344 (SEQ ID NO:67); W83144 (SEQ ID NO:68); AA110868 (SEQ ID NO:69); AV204705 (SEQ ID NO:70); AVI 17489 (SEQ ID NO:71); AV204580 (SEQ ID

NO:72); AV149555 (SEQ ID NO:73); AV217918 (SEQ ID NO:74); AV144222 (SEQ ID NO:75); AV363935 (SEQ ID NO:76); AV367917 (SEQ ID NO:77); AV131095 (SEQ ID NO:78); AA038844 (SEQ ID NO:79); AV000269 (SEQ ID NO:80); AV215700 (SEQ ID NO:81); AA574257 (SEQ ID NO:82); AI580764 (SEQ ID NO:83); AI421341 (SEQ ID NO:84); AI624271 (SEQ ID NO:85); AI673018 (SEQ ID NO:86); AI826486 (SEQ ID NO:87); AW025889 (SEQ ID NO:88); AI690995 (SEQ ID NO:89); AI934145 (SEQ ID NO:90); AI 805491 (SEQ ID NO:91); AI318424 (SEQ ID NO:92); AI694835 (SEQ ID NO:93); AI915915 (SEQ ID NO:94); AI347155 (SEQ ID NO:95); H79248 (SEQ ID

NO:96); H79154 (SEQ ID NO:97); AA883244 (SEQ ID NO:98); AI925943 (SEQ ID NO:99); AI027566 (SEQ ID NO: 100); AI422908 (SEQ ID NO: 101); H12345 (SEQ ID NO.102); H12296 (SEQ ID NO.103); AA147029 (SEQ ID NOT04); AA147030 (SEQ ID NO:105); R21923 (SEQ ID NO.106); R22572 (SEQ ID NO:107); AI264294 (SEQ ID NO:108); AI439891 (SEQ ID NO: 109);

W88749 (SEQ ID NOT 10); AI698667 (SEQ ID NOT 11); AI439894 (SEQ ID NOT 12); AI421551 (SEQ ID NOT 13); H63468 (SEQ ID NO: 114); AI082760 (SEQ ID NOT 15); W73843 (SEQ ID NOT 16); W73699 (SEQ ID NOT 17); AA535984 (SEQ ID NO: 118); AW000865 (SEQ ID NO: 119); R25346 (SEQ ID NO:120); R26538 (SEQ ID NO:121); AA936901 (SEQ ID NOT22); AI350558

(SEQ ID NO: 123); AW296973 (SEQ ID NO: 124); AI003420 (SEQ ID NO: 125); AI880806 (SEQ ID NO.126); R60563 (SEQ ID NOT27); AA356988 (SEQ ID NOT28); AL037250 (SEQ ID NOT29); AW389915 (SEQ ID NO.130); AI439880 (SEQ ID NO.131); N30591 (SEQ ID NO.132); AW242490 (SEQ ID NOT33); AI950686 (SEQ ID NOT34); AW389884 (SEQ ID NO.135);

AA374147 (SEQ IDNO:136); AW368137 (SEQ IDNO:137); AW389910 (SEQ ID NO.138); AA640616 (SEQ ID NO:139); AW079701 (SEQ ID NO.140); AI202368 (SEQ ID NO: 141); N51558 (SEQ ID NO: 142); AA401853 (SEQ ID NO: 143);AW410681 (SEQIDNO:144);N40375 (SEQIDNO:145); AW385667 (SEQ ID NO: 146); N27557 (SEQ ID NO: 147); AW368222 (SEQ ID NO: 148);

AW368132 (SEQ ID NO: 149); R60619 (SEQ ID NOT50); D20400 (SEQ ID NOT51); AW517221 (SEQ ID NO.152); AA403126 (SEQ ID NOT53).

Thus, in one embodiment the present invention is directed to polynucleotides comprising the polynucleotide fragments and full-length polynucleotide (e.g. the coding region) described herein exclusive of one or more of the above recited ESTs.

Since the plasmid containing the cDNA clone has been deposited and the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1) is provided, generating such DNA fragments would be routine to the skilled worker in the relevant art.

Restriction endonuclease cleavage or shearing by sonication, for example, may easily be used to generate fragments of various sizes. Alternatively, the DNA fragments of the present invention can be generated synthetically according to the methods and techniques known and available to those skilled in the art. The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode for fragments, analogs or derivatives of the Bridge-1 protein, e.g., polypeptides having biological activity substantially similar to the Bridge-1 protein. Variants may occur naturally, such as isoforms and allelic variants. Non-naturally occurring variants may be produced using any of the mutagenesis techniques known and available to those skilled in the art.

Such variants include those produced by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the Bridge-1 protein or fragment thereof. Also especially preferred in this regard are substitution of nucleotides that encode a conservative amino acid substitution. In a preferred embodiment, such variants contain no more than five total substitutions, deletions, and/or additions.

Further embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 80% identical, more preferably at least 85%> identical, more preferably at least 90% identical, and most preferably at least 95%, 96%, 97%, 98%, or 99% identical to: (a) the nucleotide sequence of the cDNA clone in the plasmid pcDN A3 -Bridge-1 deposited with the American Type Tissue Culture ("ATCC") and assigned accession number 203947; (b) the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1); (c) the nucleotide sequence of the cDNA clone in the plasmid pcDNA3-Bridge-l deposited with the American Type Tissue Culture ("ATCC") and assigned accession number 203947, which encodes the full-length Bridge-1 protein; (d) the nucleotide sequence of the ORF, i.e. coding region, shown in Figure 1 (SEQ ID NO: 1), which encodes the full-length Bridge-1 protein; (e) a nucleotide sequence complimentary to any of (a)-(d). Whether any two nucleic acid molecules have nucleotide sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% "identical" can be determined conventionally using known computer algorithms. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the F ASTDB computer program based on the algorithm of Brutlag et al., Comp. App. Biosci. 6:231-245 (1990). In a sequence alignment the query and subject substances are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a F ASTDB alignment of DNA sequences to calculate percent identity are:

Matrix=Unitary, k-tuple -, Mismatch Penalty=l, Joining Penalty=30, Randomization Group Length =0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter. The present application is directed to such nucleic acid molecules having a nucleotide sequence at least 90%>, 95%), 96%, 97%, 98%,

99%, identical to the nucleotide sequence of the above-recited nucleic acid molecules irrespective of whether they encode a polypeptide having Bridge-1 activity. This is because, even where a particular nucleic acid molecule encodes a polypeptide that does not have Bridge-1 activity, one of skill in the art would still know how to use the nucleic acid molecule as a probe. In at least one embodiment, the percent identity is measured by comparing the obtained DNA sequence to that of nucleotides 495-1 162 (i.e., the ORF or coding region) of the nucleotide sequence in FIG. 1 (SEQ ID NO: 1). Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having Bridge-1 activity include, wter alia, (X) isolating the Bridge-1 gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (FISH) to metaphase chromosomal spreads to provide precise chromosomal location of the Bridge-1 gene as described in Verma et al, Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988); and Northern Blot analysis for detecting Bridge- 1 mRNA expression in specific tissues, such as placenta tissue .

As used herein, "Bridge- 1 activity" is intended to mean one or more of the following: protein-protein binding activity, transcription coactivation activity, or transcription activation activity. By "transcription activation activity" is meant positive regulation of gene expression consisting of an increase in the level of transcription and/or translation resulting from interaction with core cellular transcriptional or translational machinery. By "transcription coactivation activity" is meant positive regulation of gene expression consisting of an increase in the level of transcription and/or translation resulting from interaction with other proteins with transcription activation activity. Preferred, however, are nucleic acid molecules having a nucleotide sequence at least 80%, and preferably at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of the above-described nucleic acid molecules which do, in fact, encode a polypeptide having Bridge-1 activity. As used herein, "a polypeptide having a Bridge-1 activity" is intended to mean polypeptides exhibiting similar, but not necessarily identical, activity as to the

Bridge-1 activity as measured in a particular biological assay. For example, the Bridge-1 protein of the present invention interacts directly with known transcription factors such as PDX-1, E12 and E47. Moreover, when recombinantly expressed in mammalian cells, the Bridge-1 protein of the present invention enhances transcription genes modulated by these transcription factors. Thus, "a polypeptide having a Bridge-1 protein activity" includes polypeptides that interact with PDX-1, El 2, E47 and other transcription factors or otherwise enhance PDX- 1 or E2A protein dependent transcriptional activation or PDX- 1 or E2A independent transcription activation. Due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a nucleotide sequence at least 90%>, preferably at least 95%), 96%), 97%, 98%o, 99%) identical to the nucleotide sequence of the above-described nucleic acid molecules will encode "a polypeptide having Bridge-1 activity." In fact, since degenerate variants all encode the same polypeptide, this will be clear to the skilled artisan. It will be further recognized by those skilled in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having Bridge-1 activity. This is because the skilled artisan is fully aware of possible amino acid substitutions that are either less likely or not likely to significantly affect protein function (e.g. , replacing one aliphatic amino acid with a second aliphatic amino acid).

Guidance concerning how to make phenotypically silent amino acid substitutions is provided, for example, in J.U. Bowie et al, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247. 1306-1310 (1990), the entire contents of which is hereby incorporated by reference herein, wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality. As the authors state, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie et al, supra, and the references cited therein.

Vectors and Host Cells

The present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of Bridge-1 or Bridge-1 fragments. As used herein, "Bridge-1 fragment" means a shortened sequence of an amino acid sequence that retains some or all of the the Bridge-1 activity of the full-length sequence, such as a fragment comprising the Bridge-1 PDZ-like domain (amino acids 138-178 of the amino acid sequence shown in FIG.

1 (SEQ ID NO: 2)), the Bridge-1 carboxyl terminus domain (amino acids 186-222 of the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2)), or the Bridge-1 PDZ-like domain and the Bridge-1 carboxyl terminus domain (amino acids 138- 222 of the amino acid sequence shown in FIG. 1 (SEQ ID NO:2)). The term "Bridge-1 fragment" is also intended to refer to splice-variants and proteolytic fragments of the full-length Bridge-1 amino acid sequence shown in FIG. 1 (SEQ ID NO:2), including the "small form" Bridge-1 (FIG. 17) which migrates at approximately 18 kD in SDS-PAGE Western Blots, is detected with rabbit polyclonal Bridge-1 antisera, and is differentially expressed in cell lines derived from different tissues with preferential expression in pancreatic beta cells..

Recombinant constructs may be introduced into host cells using well known techniques such as infection, transduction, transfection, transvection, electroporation and transformation. The vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

Preferred are vectors comprising cis-acting control regions to the polynucleotide of interest. Appropriate trans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide for specific expression, which may be inducible and/or cell type-specific. Particularly preferred among such vectors are those inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives.

Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as cosmids and phagemids.

The DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters, CMV promoters, promoters of retroviral LTRs, and inducible promoters such as tetracycline and IPTG inducible promoters as well as promoters inducible with heavy metals to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating AUG at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated. As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as Ε. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, Cos and

Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Illustrative examples of vectors preferred for use in bacteria include, but are not limited to, pA2, pQΕ70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Preferred eukaryotic vectors include, but are not limited to, pcDNA-3 (Invitrogen), pM, pVP16 (Clonetech), pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

Among known bacterial promoters suitable for use in the present invention include the E. coli lad and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV"), and metallothionein promoters, such as the mouse metallothionein-I promoter.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Ausubel, F. M. et al, CURRENTPROTOCOLSINMOLECULARBIOLOGY, (John Wiley and Sons, Inc.) 1994- 1997. Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, generally about 10 to 300 bp in size, that act to increase transcriptional activity of a promoter in a given host cell-type. Illustrative examples of enhancers include, but are not limited to, the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals. The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification.

The Bridge-1 protein or fraction thereof can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.

Polypeptides of the present invention include, but are not limited to, naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be post translationally modified (e.g., glycosylated, phosphorylated, famesylated, etc.). In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

Bridge-1 Polypeptides and Fragments

The invention further provides an isolated Bridge- 1 polypeptide having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence as shown in Figure 1 (SEQ ID NO:2), or a fragment thereof. Preferred Bridge-1 fragments will have Bridge-1 activity. Preferred Bridge-1 fragments should at least include amino acid residues 138 to 178 as shown in FIG.l (SEQ ID NO:2) and/or amino acid residues 186-222 as shown in FIG. 1 (SEQ ID NO:2), or amino acid substitutions, additions or deletions thereof that are not significantly detrimental to Bridge-1 activity.

Bridge-1 polypeptide fragments may be "free-standing" or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Polypeptide fragments may comprise 9, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more amino acids in length.

In one embodiment, the present invention is directed to Bridge-1 polypeptides and polypeptide fragments described herein exclusive of one or more of the polypeptides described in SEQ ID NOS: 3-20.

As used herein, an "isolated" polypeptide or protein is intended to mean a polypeptide or protein removed from its native environment, such as recombinantly produced polypeptides and proteins expressed in host cells and native or recombinant polypeptides which have been substantially purified by any suitable technique (e.g. , the single-step purification method disclosed in Smith and Johnson, Gene <57/31-40 (1988), which is incorporated by reference herein). Isolated polypeptides or proteins according to the present invention further include such compounds produced synthetically. The inventors have discovered that the full-length Bridge-1 protein is an about 222 amino acid residue protein with a deduced molecular weight of about 24.8 kD and a pi of 6.70. It will be recognized by those skilled in the art that some of the amino acid sequence of the Bridge-1 protein can be varied without significant effect on the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity, such as the PDZ-like domain and the carboxyl terminus domain described above which have been determined by the inventors as being critical to Bridge-1 activity. In general, it is often possible to replace residues which form the tertiary structure, provided that residues performing a similar function are used. In other instances, the type of residue may be completely unimportant if the alteration occurs at a non-critical region of the protein.

Thus, the present invention further includes variations of the Bridge-1 polypeptide which show substantial Bridge- 1 polypeptide activity or which include regions of Bridge-1 protein. Such mutants include deletions, insertions, inversions, repeats, and type substitutions (for example, substituting one hydrophilic residue for another, but not strongly hydrophilic for strongly hydrophobic as a rule). Small changes or such "neutral" amino acid substitutions will generally have little effect on activity. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe and Tyr. As indicated in detail above, further guidance concerning which amino acid changes are likely to be phenotypically silent (i.e., not likely to have a significant deleterious effect on a function) can be found in Bowie et al, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247. 1306-1310 (1990), the entire contents of which is hereby expressly incorporated herein by reference.

The polypeptides of the present invention include polypeptides having an amino acid sequence as encoded by the deposited cDNA, an amino acid sequence as shown in FIGURE 1 (SEQ ID NO:2), as well as an amino acid sequence at least 80%> identical, more preferably at least 85%> identical, more preferably at least 90%> identical, and most preferably at least 95%, 96%, 97%, 98%, or 99% identical, to the amino acid sequence encoded by the deposited cDNA, to the amino acid sequence as shown in FIGURE 1 (SEQ ID NO:2), or to the amino acid sequence of a polypeptide fragment described above. Whether two polypeptides have an amino acid sequence that is at least 80%>, 90%o or 95%> identical can be determined using a computer algorithm as described above.

As described in detail below, the nucleic acid molecules and polypeptides of the present invention are useful in screening assays for identifying proteins and protein fragments that bind to Bridge- 1 or a Bridge- 1 fragment, including proteins, protein fragments, biological and chemical compounds and other small molecules that enhance or inhibit Bridge- 1 activity. Accordingly, the nucleic acid molecules and polypeptides of the present invention are useful in assays for identifying drugs capable of enhancing or inhibiting Bridge-1 activity.

Fusion Proteins

The full-length Bridge- 1 protein, and Bridge- 1 polypeptide fragments can be used to generate fusion proteins. For example, the Bridge- 1 polypeptide, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the Bridge-1 polypeptide can be used to indirectly detect the second protein by binding to the Bridge-1 protein. Moreover, the Bridge-1 polypeptides can be used as a targeting molecule once fused to other proteins.

Examples of domains that can be fused to Bridge-1 polypeptides include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.

In certain preferred embodiments, Bridge-1 fusion polypeptides may be constructed which include additional N-terminal and/or C-terminal amino acid residues. In particular, any N-terminally or C-terminally deleted Bridge-1 polypeptide disclosed herein may be altered by inclusion of additional amino acid residues at the N-terminus to produce a Bridge-1 fusion polypeptide. In addition, Bridge-1 fusion polypeptides are contemplated which include additional N-terminal and/or C-terminal amino acid residues fused to a Bridge- 1 polypeptide comprising any combination of N- and C-terminal deletions. Moreover, fusion proteins may also be engineered to improve characteristics of the Bridge-1 polypeptide. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the Bridge-1 polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the Bridge- 1 polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the Bridge-1 polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.

Moreover, Bridge-1 polypeptides, including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins

(IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A394,827; Trauneckeretα/.,Nαtwre 337:84-86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et α/., J. Biochem. 270:3958-3964 (1995).) Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 5:52-58

(1995); K. Johanson et α/., J. Biol. Chem. 270:9459-941 (1995).)

Moreover, the Bridge-1 polypeptides can be fused to marker sequences, such as a peptide which facilitates purification of Bridge-1. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,

Chatsworth, CA, 9131 1), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci. USA 5(5:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein.

(Wilson et al, Cell 37:161 (1984).)

Thus, any of these above fusions can be engineered using the Bridge-1 polynucleotides or the Bridge-1 polypeptides. Screening Methods

As indicated, the inventors have cloned a gene encoding Bridge- 1 and have shown that the full length Bridge-1 protein and Bridge-1 fragments bind other proteins. Further, the inventors have shown that Bridge-1 proteins and protein fragments interact with other transcription factors, including members of the E2A family of transcription factors (which includes the El 2 E47, E2-2, HEB and daughterless transcription factors), PDX-1 and others, to modulate transcription activation. Thus, the present invention further provides for screening methods for identifying proteins, protein fragments, biological and chemical compounds, and other small molecules that interact with Bridge-1 or Bridge-1 fragments. Such screening methods are useful for identifying, proteins, protein fragments, biological and chemical compounds and molecules that enhance or inhibit Bridge- 1 activity. In general, such methods involve: (a) providing a host cell containing recombinant genes which express a polypeptide comprising a protein ligand binding domain and a polypeptide comprising Bridge- 1 or a Bridge- 1 fragment, or fusion proteins comprising a protein ligand binding domain and a polypeptide comprising Bridge-1 or a Bridge-1 fragment, wherein said Bridge-1 and said Bridge- 1 fragment bind said protein ligand binding domain; (b) administering a candidate polypeptide to said cell; and (c) determining whether said candidate polypeptide reduces either: (1) Bridge-1 or Bridge-1 -fragment-binding to the polypeptide protein binding domain as compared to said binding in the absence of said candidate polypeptide; or (2) Bridge-1 or Bridge-1 -fragment-transcription coactivation activity ; or (3 ) Bridge- 1 or Bridge- 1 fragment transcription activation activity as compared to such activities in the absence of said candidate polypeptide. Such screening methods are known in the art. (Ausubel, F. M. et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Chapter 20 (John Wiley & Sons Inc.) (1994-1997); Coligan, J. E. et al, CURRENT PROTOCOLS IN PROTEIN SCIENCE, Chapter 19 (John Wiley & Sons, Inc.) (1995-1997)). A preferred assay uses the mammalian two-hybrid system (Luo, Y. et al, Biotechniques 22:350-352 (1997) (the entire contents of which is herein expressly incorporated by reference. These methods can used to screen for transcription factor-coactivator interactions as described in WO 98/45423, the entire contents of which is herein fully incorporated by reference. Recombinant genes encoding a polypeptide comprising Bridge-1 or a

Bridge-1 fragment capable of binding other proteins are described above. Methods for determining whether a candidate protein enhances or interferes with Bridge-1 or Bridge-1 -fragment binding are known in the art. For example, the effect of a candidate protein on Bridge- 1 or Bridge- 1 -fragment-binding to another protein, for example a protein in the E2A transcription factor or PDX-1 , can be studied using glutathione-S-transferase (GST) interaction assays. Alternatively, this determination could be made using the mammalian two-hybrid system (Luo, Y. et al., Biotechniques 22:350-352 (1997).

The inventors have identified at least two domains of Bridge- 1 , a PDZ-like domain (amino acids 138- 178 of the amino acid sequence shown in FIG. 1 (SEQ

ID NO:2)), and a carboxyl terminus domain (amino acids 186-222 of the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2), which mediate Bridge-1 activity. Further, the inventors have shown that polypeptides containing these domains, when fused to a DNA-binding domain of a transcriptional activator, are capable of activating transcription. Accordingly, the present invention further provides a screening method for identifying a polypeptide which binds to the Bridge- 1 PDZ- like domain and/or the Bridge-1 carboxyl terminus domain which comprises (a) providing a host cell containing a recombinant gene or genes which express a polypeptide comprising a transcriptional activator DNA-binding domain (DBD) and the Bridge- 1 PDZ-like domain and/or the Bridge- 1 carboxyl terminus domain;

(b) administering a recombinant gene expressing a candidate polypeptide or a biological or chemical compound or small molecule to said cell; and (c) determining whether said candidate polypeptide, biological or chemical compound or small molecule enhances Bridge-1 activity. The invention further provides for a screening method for identifying polypeptides that bind to the Bridge-1 PDZ-like domain and/or the Bridge-1 carboxyl terminus domain, which comprises: (a) providing a host cell containing a recombinant gene or genes which express a polypeptide comprising a transcriptional activator DNA-binding domain (DBD) and a Bridge-1 PDZ-like domain and/or Bridge-1 carboxyl terminus domain; (b) administering a recombinant gene expressing a candidate polypeptide or a biological or chemical compound or other small molecule to said cell; and (c) determining whether said candidate polypeptide, biological or chemical compound or other small molecule inhibits Bridge-1 activity.

By "transcriptional activator" it is meant molecules that enhance transcription by RNA polymerase B (II). Transcriptional activators include yeast transcriptional activators, such as GAL4 and GCN4; the herpes simplex activator, VP16; and members of the nuclear receptor family, which includes RAR, RXR, ER, TR, NDR, GR, and AR.

Recombinant genes encoding a polypeptide comprising a Bridge-1 PDZ- like domain are described below. Recombinant genes encoding a polypeptide comprising a transcriptional activator DBD are well known in the art. Methods for determining whether a candidate polypeptide enhances or interferes with transcription are well known in the art. For example, the effect of a candidate polypeptide on Bridge- 1 PDZ-like domain activity or Bridge- 1 carboxyl terminus domain activity can be determined using CAT assays as described below and in Gronemeyer et al. (1987) and Bocquel et al, Nucl. Acids Res. (1989).

Where the effect of a candidate polypeptide is to be determined, preferably, recombinant genes will encode a chimeric polypeptide comprising a transcriptional activator DBD fused to a Bridge- 1 polypeptide comprising the PDZ-like domain. In a further embodiment, the host cell expressing the recombinant genes will also express a reporter gene. Examples of reporter genes are described above. Bridge-1 Antibodies and Methods of Use

Bridge-1 antibodies are also provided by the present invention, as specific for a Bridge-1 protein or a Bridge-1 protein fragment. The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments thereof provided by any known technique, such as, but not limited to enzymatic cleavage, peptide synthesis or recombinant techniques. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen. A monoclonal antibody contains a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites. MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature 256:495-497 (1975); U.S. Patent No. 4,376,110; Ausubel et al, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Assoc. and Wiley Interscience,

N.Y., (1987-1996); and Harlow and Lane ANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Laboratory (1988); Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N. Y., ( 1992- 1996), the contents of which references are incorporated entirely herein by reference.

Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof. A hybridoma producing a mAb of the present invention may be cultivated in vitro, in situ or in vivo. Production of high titers of mAbs in vivo or in situ makes this the presently preferred method of production.

Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having variable region derived from a murine mAb and a human immunoglobulin constant region, which are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (Cabilly et al, Proc. Natl. Acad. Sci. USA 81 :3273-3277 (1984); European Patent Application 125023 (published November 14, 1984); Neuberger et al., Nature

314:268-270 (1985); Taniguchi et al, European Patent Application 171496 (1985); Morrison et al, European Patent Application 173494 (1986); Neuberger et al, PCT Application WO 86/01533, (1986); Kudo et al, European Patent Application 184187 (1986); Morrison etal., European Patent Application 173494 (1986); Robinson et al., PCT Publication PCT/US86/02269 (1987); Liu et al,

Proc. Natl. Acad. Sci. USA 54:3439-3443 (1987); Sun et al, Proc. Natl. Acad. Sci. USA 54:214-218 (1987); Better et al, Science 240:1041- 1043 (1988); and Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Laboratory (1988)). These references are entirely incorporated herein by reference.

An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., mouse strain) as the source of the mAb with the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). See, for example, U.S. patent No. 4,699,880, which is herein entirely incorporated by reference.

The anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. The anti-anti-Id may be epitopically identical to the original mAb which induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies of identical specificity. The anti-Id mAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as GRB protein- .

The term "antibody" is also meant to include both intact immunoglobulin molecules as well as fragments thereof, such as, for example, Fab and F(ab')₂, which are capable of binding antigen. Fab and F(ab')₂ fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). It will be appreciated that Fab and F(ab')₂ and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of a Bridge- 1 according to the methods disclosed herein for intact antibody molecules. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')₂ fragments).

An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.

An "antigen" is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one, or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

The antibodies, or fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect a Bridge-1 protein, polypeptide, or fragment, in a sample or to detect presence of cells which express a Bridge-1 of the present invention. This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorometric detection. The antibodies (or fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of a Bridge-1 protein, polypeptide, or fragment, of the present invention. In situ detection may be accomplished by removing a histological specimen form a patient, and providing a labeled antibody of the present invention to such a specimen. The antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of a Bridge-1 protein, polypeptide, or fragment, but also its distribution on the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Assays for a Bridge-1 protein, polypeptide, or fragment, of the present invention typically comprises incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leukocytes, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying a Bridge-1 protein, polypeptide, or fragment, and detecting the antibody by any of a number of techniques well- known in the art. The biological sample may be treated with a solid phase support or carrier such as nitrocellulose, or other solid support or carrier which is capable of immobilizing cells, cell particles or soluble proteins. The support or carrier may then be washed with suitable buffers followed by treatment with a detectably labeled Bridge-1 -specific antibody. The solid phase support or carrier may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on said solid support or carrier may then be detected by conventional means.

By "solid phase support", "solid phase carrier", "solid support", "solid carrier", "support" or "carrier" is intended any support or carrier capable of binding antigen or antibodies. Well-known supports or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support or carrier configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports or carriers include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-Bridge- 1 antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation. Other such steps as washing, stirring, shaking, filtering and the like may be added to the assays as is customary or necessary for the particular situation.

One of the ways in which a Bridge- 1 -specific antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5 -steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may be accomplished using any of a variety of other immunoassays. For example, by radioactivity labeling the antibodies or antibody fragments, it is possible to detect R-PTPase through the use of a radioimmunoassay (RIA). A good description of RIA maybe found in Laboratory Techniques and Bio chemistry in Molecular Biology, by Work, T.S. et al., North Holland Publishing Company, NY (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., incorporated by reference herein. The radioactive isotope can be detected by such means as the use of a γ counter or a scintillation counter or by autoradiography .

It is also possible to label an anti-Bridge- 1 antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can be then be detected due to fluorescence.

Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals such as those of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (EDTA).

The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin. An antibody molecule of the present invention may be adapted for utilization in a immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or carrier and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.

Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen form the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support or carrier is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support or carrier through the unlabeled antibody, the solid support or carrier is washed a second time to remove the unreacted labeled antibody.

In another type of "sandwich" assay, which may also be useful with the antigens of the present invention, the so-called "simultaneous" and "reverse" assays are used. A "simultaneous" and "reverse" assays are used. A simultaneous assay involves a single incubation step as the antibody bound to the solid support or carrier and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support or carrier is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support or carrier is then determined as it would be in a conventional "forward" sandwich assay.

In the "reverse" assay, stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support or carrier after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support or carrier is then determined as in the "simultaneous" and "forward" assays.

Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

Examples

Example 1 Materials and Methods

Cloning of Rat Bridge by a Yeast Two-hybrid System

Standard molecular biology techniques were used (Sambrook, J., et al, "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY: (1989)). A directional INS- 1 cDNA library was constructed in the plasmid vector pJG4-5 using a Stratagene cDNA synthesis kit. The El 2 bait was constructed by RT-PCR amplification of DNA encoding amino acids 521 to 649 of El 2 from total RNA isolated from rat 18 dpc pancreas, followed by cloning into the plasmid pEG202 in frame with an upstream LexA DNA-binding domain. Yeast two-hybrid screening was conducted according to standard methods (Golemis, E. A., in Current Protocols in Molecular Biology, F.M. Ausubel, et al, eds., John Wiley and Sons, New York. pp. 13.14.1-13.14.17 (1994)). The complete 1.4 kb Bridge-1 cDNA was isolated by the screening of a rat 14 dpc pancreatic library using the 30-mer oligonucleotide 5'-TCACTCGACATCGCGGACCTAGCCTAAAA-3' (SEQ ID NO:21). Sequencing was performed by the Sanger dideoxy chain termination method. Protein similarity indices were determined using the Lipman-Pearson Protein Alignment function of the software package Lasergene (DNASTAR, Inc.,

Madison, WI) and the alignment program Blast (Genbank). Rat Bridge- 1 cDNA sequence was submitted to Genbank under the accession number AF067728.

Northern Blot Analysis

Total RNAs were extracted by the guanidinium isothiocyanate method (Davis, L. G., etal, Basic Methods in Molecular, New York, Elsevier( 1986), pp.

129- 135) and poly (A)+ RNA was prepared using the PolyATract mRNA isolation system (Promega, Madison, WI). RNAs were electrophoresed on 1%> agarose-formaldehyde gels and blotted onto nylon membranes (GeneScreen, NEN Life Science Products, Boston, MA) prior to probing with ³²P-labeled Bridge-1 cDNA. Membranes were stripped and reprobed with rat γ-actin according to the manufacturer's instructions. Mouse and human endocrine system multiple tissue Northern blots (Clontech Laboratories Inc., Palo Alto, CA) were probed with ³²P-labeled Bridge-1 cDNA using high stringency washing conditions as described by the manufacturer. Plasmid Construction

pBSII-Bridge-1 and pcDN A3 -Bridge-1 were constructed by inserting the 1.4 kb Bridge-1 cDNA into an EcoRI site within the multiple cloning region of pBSII or pcDNA3, respectively. For the mammalian two-hybrid studies, the Mammalian Matchmaker Two-Hybrid Assay Kit (Clontech Laboratories Inc., Palo

Alto, CA) vectors pM and PNP 16 were used to construct plasmids expressing GaW DΝA-binding domain/Bridge- 1, NP16/Bridge-l , GaW DΝA-binding domain/E12, NP16/E12, and GaW DΝA-binding domain/Beta-2 fusion proteins. pM-Bridge-1 and pNP16-Bridge-l were constructed by inserting a blunt-ended 900 bp BstU I/EcoRI fragment of pBSII-Bridge- 1 in frame into a blunt-ended Mlu

I site of the multiple cloning site of the plasmids pM and pNP16, respectively. pM-E12 and pNPl 6-E 12 were constructed by inserting a 2.7 kb blunt-ended Νde I/EcoRI fragment of Pan 2 excised from the vector PARP5 (gift of C. Nelson) in frame into a Sma I site of pM and pNP16 vectors, respectively. pM-Beta-2 was constructed by inserting a 2.4 kB blunt-ended BamHI/cohesive-ended Xba I fragment from pcDΝAl-Beta-2 (gift of J. Seufert) into the vector pM, that had been prepared by digesting with BamHI, followed by blunting with Klenow polymerase and digesting with Xba I. pcDΝAI-Beta-2 had been previously constructed by inserting a 2.4 kB BamHI/Xho I fragment from pCMN-Beta-2 (gift of F. Νaya and M.J. Tsai) into pcDΝAI that had been digested with BamHI and

Xho I. After cloning was completed, the matchmaker vector constructs were verified by automated DΝA sequencing. pcDΝA3-E12 and pcDNA3-E47 were constructed by inserting 2.7 kb Bgl II/EcoRI fragments of Pan 2 from the vector PARP5 or Pan 1 from the vector PARP5P2, respectively, (vectors were gifts of C. Nelson) into pcDNA3 prepared by digestion with BamHI and EcoRI.

5FF 1 CAT was a gift from J. L. Moss. pcDNA3-E12ΔC and pVP16-E12ΔC were constructed by point mutagenesis with insertion of a premature stop codon in pcDNA3 -E 12 and pVP 16-E 12, respectively, using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer's i n s t r u c t i o n s w i t h t h e o l i g o n u c l e o t i d e s 5'-ACCCGGGCCTGGGTTAGGCCCACAAT-3'(SEQ ID NO: 22) and 5 ' - ATTGTGGGCCTAACCCAGGC-CCGGGT-3 ' (SEQ ID NO: 23). pM-Bridge-l(l-72) and pM-Bridge-l(l-184) were constructed by point mutagenesis with insertion of premature stop codons in pM-Bridge- 1 , utilizing the

QuikChange Site-Directed Mutagenesis Kit. The oligonucleotides used for construction of pM-Bridge-l(l-72) were

5'-GGATTTGTATCAGGTCTGAACAGCAAGGCAC-3' (SEQIDNO: 24) and 5 ' -GTGCCTTGCTGTTC AGACCTGATAC AAATCC-3 ' f SEQ IDNO:25) and of pM-Bridge- 1(1-184) were

5 ' -CAGCAC AGCGAGGGGTAGCCCCTGAATGTC-3 ' (SEQ ID NO: 26) and 5 '-GACATTCA-GGGGCTACCCCTCGCTGTGCTG-3 ' (SEQ ID NO: 27). pM-Bridge- 1(1-133) was constructed by digestion of pM-Bridge- 1 with Stu I and Hind III, blunting with Klenow polymerase, and religation. Mutants were verified by sequencing and expression was assessed by Western blotting of transfected cell extracts.

Cell Culture and Immunocytochemistry

HeLa cells (American Type Culture Collection, Manassas, VA, and gift from R. Stein), BHK-21 (C-13) cells (American Type Culture Collection, Manassas, VA), and RIN1027-B2 cells (Philippe, J., J. Clin. Invest. 79:351-358

(1987)) were grown in Dulbecco's Modified Eagle Medium (4.5 g of glucose per liter) supplemented with 10%) fetal bovine serum, 100 U of penicillin G and 100 μg streptomycin sulfate per ml (GIBCO BRL Life Technologies, Inc., Gaithersburg, MD). For immunocytochemistry, RIN1027-B2 cells were grown on glass slide culture chambers (Nunc, Inc., Naperville, IL) prior to staining.

Slides were rinsed several times in phosphate-buffered saline (PBS) followed by fixation in 4% paraformaldehyde in PBS for 10 minutes at room temperature. After several additional rinses in PBS, cells were permeabilized with 100% methanol at -20°C for 5 minutes followed by blocking with 1% normal donkey serum for 20 minutes at room temperature. Slides were then incubated with preimmune or rabbit polyclonal anti-Bridge- 1 antisera generated against the peptide immunogen EEALHQLHARDKEKQ (SEQ ID NO: 28) at a dilution of 1 :500 at 4°C overnight. After several additional rinses in PBS, cells were incubated with donkey anti-rabbit IgG Cy2 (Jackson Immuno Research Laboratories, West Grove, PA) at a dilution of 1 :500 for 1 hour at room temperature in the dark. Slides were then rinsed with PBS and mounted with fluorescence mounting medium (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Adult murine pancreas was embedded in Tissue-Tek O.C.T. compound

(Sakura Finetek, Torrance, CA) and frozen on dry ice. Tissue was sectioned at 7 micron increments and fixed in 4% paraformaldehyde in PBS for 10 minutes at room temperature. After 4 rinses with PBS, permeabilization with methanol, and blocking with 1%> donkey serum, sections were incubated overnight at 4°C either with rabbit polyclonal anti-Bridge-1 antiserum (1 :500 dilution) and guinea pig anti-insulin antiserum (1 :200 dilution) (Linco Research, Inc., St. Charles, MO) or with preimmune rabbit serum (1 :500 dilution) and guinea pig anti-insulin antiserum (1 :200 dilution). Sections were then rinsed in PBS and incubated 90 minutes with donkey anti-rabbit IgG Cy3 ( 1 : 1500 dilution) and donkey anti-guinea pig IgG Cy2 (1 :500 dilution) (Jackson Immuno Research Laboratories, West

Grove, PA). Sections were then rinsed in PBS and mounted in fluorescence mounting medium (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Embryonic day 19 mouse pancreas was fixed in 4% paraformaldehyde, followed by incubation in 30%) sucrose prior to embedding in paraffin. Sections were cut at 5 micron intervals and paraffin was extracted with sequential washes in xylene, ethanol solutions, and phosphate-buffered saline. Immunostaining was conducted with rabbit polyclonal anti-Bridge-1 antisera (1 :500 dilution). Slides were incubated with a biotinylated secondary antibody followed by an avidin-biotinylated horseradish peroxidase complex (Vectastain ABC System, Vector Laboratories, Burlingame, CA). Images were acquired with a Nikon Epifluorescence microscope with an Optronics TEC-470 camera (Optronics Engineering, Goleta, CA) with an interface to a Power Macintosh 7100 computer. Image analysis was conducted with IP Lab Spectrum software (Signal Analytics Corp., Vienna, VA) and Adobe Photoshop 4.0 software (Adobe Systems Incorporated, San Jose, CA).

Transfections

In some experiments HeLa cells were transfected with 25 ug total DNA by the calcium phosphate precipitation method using the CalPhos Maximizer Transfection Kit (Clontech Laboratories, Inc., Palo Alto, CA) according to the manufacturer' s instructions. In additional studies BHK cells or HeLa cells were transfected with 5 μg total DNA and 5 μl Lipofectamine as outlined by the manufacturer (GIBCO BRL Life Technologies, Inc., Gaithersburg, MD). Cells were harvested 48 hours after transfection. Chloramphenicol acetyltransferase (CAT) assays were conducted with the fluorescent substrate assay kit FAST CAT (Molecular Probes, Eugene, OR) and thin layer chromatography on silica gel plates (Eastman Kodak, Rochester, NY) as previously reported (Staffers, D. A., et al, J. Clin. Invest. 702:232-241 (1998)). Quantitation was performed with a Fluorimager 575 interfaced with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Luciferase assays were conducted as previously described (Lu, M. et al, J. Biol. Chem. 272:28349-28359 (1997)).

In Vitro Transcription and Translation Reactions

Rat Bridge- 1 was synthesized in rabbit reticulocyte extracts by coupled in vitro transcription and translation from the plasmid pcDNA3 -Bridge- 1 , using the

T7 polymerase and the TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI), according to the manufacturer's instructions. In vitro translated

E12 and E47 were generated using the same procedure with the plasmids pcDNA3-E12 and pcDNA3-E47, respectively. Reactions were conducted with either cold or ³⁵S-radiolabeled methionine (NEN Life Science Products, Boston, MA). To visualize the incorporation of ³⁵S-methionine, reactions were subjected to 10% SDS-polyacrylamide electrophoresis, followed by gel incubation in an autoradiography enhancer (Enlightening, NEN Life Science Products, Boston,

MA) prior to autoradiography.

Western Blot Analysis

In vitro translated reaction products were fractionated by 10%> SDS-polyacrylamide electrophoresis (SDS-PAGE), electroblotted onto Immobilon-P membranes (Millipore, Bedford, MA) and incubated with rabbit polyclonal anti-Bridge- 1 antisera ( 1 :2000 dilution). Extracts from transfected cells were fractionated by SDS-PAGE, electroblotted onto Immobilon-P membranes and incubated with rabbit polyclonal anti-Bridge-1 antisera (1:2000 dilution), rabbit polyclonal anti-GaWDBD antiserum (1 : 1000 dilution) (sc-577, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or rabbit polyclonal anti-El 2 antisera

(1 : 1000 dilutions) (sc-349 and sc-762, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Protein bands were visualized by chemiluminescence with ECL Western blotting detection reagents (Amersham Life Sciences, Arlington Heights, IL) using a horseradish peroxidase-conjugated goat anti-rabbit antibody (Bio-Rad Laboratories, Richmond, CA).

Immunoprecipitations

In vitro translated proteins were pre-incubated in phosphate-buffered saline for 30 minutes at 4°C prior to preclearing with Protein A Sepharose (Pharmacia

Biotech AB, Uppsala, Sweden) for 30 additional minutes. Protein supernatants were incubated for one hour at 4°C with rabbit polyclonal anti-Bridge-1 antisera or preimmune antisera. Immune complexes were then precipitated with Protein A Sepharose, washed, and separated by SDS-polyacrylamide electrophoresis, followed by autoradiographic enhancement and autoradiography.

Example 2 Cloning and Characterization of Bridge-1

To identify proteins that might modulate the activity of E2A on target genes in pancreatic β-cells, such as the insulin gene, a yeast two-hybrid screening system was developed. A cDNA library derived from the insulinoma cell line, INS-1 (Asfari, M., et al, Endocrinol. 730:167-178 (1992)), was screened using a bait derived from the carboxy terminus of El 2 that included the bHLH DNA-binding and dimerization domains . Approximately 0.5 X 10⁶ colonies were screened to identify four clones that interacted strongly with El 2 (Table 1). Sequencing and database comparisons identified clones #169 and #6 as rat twist and Id3, respectively; both proteins are class B bHLH proteins known to dimerize with E12 (Loveys, D. A., etal, Nucleic Acids Res. 24:2813-2820 (1996); Spicer, D. B., etα/., Twist. Science 272: 1476- 1480 (1996)). Their isolation indicated that the E12 bait worked as predicted in the yeast two-hybrid screening system. In addition, two novel sequences were identified. Clone #36 encodes a novel 177-amino acid open reading frame with homology to PDZ domains (Yao and Wong, unpublished data) and clone #18 encodes Bridge- 1 , which is the focus of this application. Interactions of these four clones with unrelated baits, including human interleukin receptor (cytoplasmic domain, amino acids 477 to 527) and D. melanogaster bicoid (pRFHM-1) (Golemis, E. A., in Current Protocols in Molecular Biology, F.M. Ausubel, et al, eds., John Wiley and Sons, New York, pp. 13.14.1-13.14.17 (1994)) were tested as negative controls. Clone #18 contained a cDNA insert of 934 bp. Because Northern blot analysis of INS-1 RNAs revealed two larger Bridge-1 transcripts of 1.3 and 1.0 kb (FIG. 3a), a 14 dpc rat pancreatic cDNA library was screened, using a 30-mer oligonucleotide from the clone #18 sequence. A single clone (Bridge-1) with an insert of 1.4 kb was isolated (FIG. 1). DNA sequence analysis revealed an open reading frame of 222 amino acids. The start codon for this open reading frame lies within the yeast two-hybrid clone #18.

Table 1 Specificity of Clone Interactions with E12 in Yeast Two-hybrid Screening

IL-R Bicoid E12

Clone

Identification Number

169 rat twist - - ++ 6 rat ld3 - - ++

36 PIN-1 - - ++

18 Bridge-1 - - ++

Four strong El 2 interacting clones identified by yeast two-hybrid screening were tested for their interaction with human interleukin-1 receptor (IL-R), D. melanogaster bicoid, and rat E12 fusion proteins by semi-quantitative yeast two-hybrid interaction assay. Strength of interaction was measured by growth on leucine dropout plates to follow LEU reporter gene activity and by the intensity of blue color on X-gal plates to assess lacZ reporter gene activity, after 30°C incubation for 72 hours. - denotes no growth on leucine dropout plates and white colonies on X-gal plates. ++ indicates growth on leucine dropout plates and intense blue colonies on X-gal plates.

The Bridge-1 sequence of 222 amino acids predicts a protein with a molecular mass of 24.8 kD and a pi of 6.70. When Bridge cDNA was introduced into a coupled in vitro transcription/translation system under the control of a T7

RNA polymerase promoter, a single radiolabeled protein was produced that migrated at approximately 28-29 kD (FIG. 3d).

Comparison of the rat Bridge-1 protein sequence with the Genbank database via Blast analysis revealed that Bridge-1 is highly conserved across species, including C. elegans, and S. cerevisiae (FIG. 2A). Bridge-1 cDNA is homologous to murine and human expressed sequence tag sequences from a variety of embryonic and adult tissues as well as to a human sequence with the designation proteasomal modulator subunit p27 (Genbank ace. no. AB003177). Rat Bridge-1 and human p27 are highly homologous, with 70 percent identity (156 of 222 amino acids) and 82 percent similarity at the protein level. The two sequences diverge at the carboxy termini of the proteins. Comparison of the first

184 amino acids of rat Bridge-1 and p27 proteins yields 84 percent identity and 98 percent similarity. Of note predicted translations of several human expressed sequence tagged sequences in the Genbank database diverge from the p27 sequence at the carboxy terminus and more closely resemble the carboxy terminus of rat Bridge-1 (Weinman, E. J., et al, J. Clin. Invest. 95:2143-2149 (1995)).

Homologies between Bridge-1 and proteins from S. cerevisiae and C. elegans are weaker, with 37 and 35 percent identity, respectively.

Example 3 Bridge-1 Has a PDZ-like Domain

Further analysis of the Bridge- 1 protein sequence revealed a 41 amino acid segment of Bridge- 1 , extending from amino acids 138 to 178, that is homologous to protein-protein interaction domains of the PDZ type within several other proteins (FIG. 2B). Homologies with the aligned proteins within this segment range from 27 to 54 percent identity and 46 to 77 percent similarity. This segment of homologous sequence in Bridge- 1 is shorter than prototypical PDZ domains of approximately 80 to 90 amino acids that form 5 or 6 beta sheets and two alpha helices by crystal structure analysis (Cabral, J. H. M., etal, Nature 3352:649-652 (1996); Doyle, D. A. et al, Cell 55:1067-1076 (1996)). The high degree of sequence similarity in this region indicates that Bridge-1 contains a PDZ-like domain. A comparison of a longer segment of Bridge-1 sequence with typical

PDZ domains from the proteins PSD-95, DLG- 1 , and ZO- 1 demonstrates that the 41 amino acid segment identified within Bridge-1 corresponds to 3 beta sheets (βC-βE) and 2 alpha helices (αA and B) of PDZ domains (FIG. 2C). The distance between beta sheets B and C varies among PDZ domains within different proteins (Cabral, J. H. M., et al, Nature 3352:649-652 (1996); Doyle, D. A. et al, Cell 55:1067-1076 (1996)), raising the possibility that additional upstream sequences within Bridge-1 may contribute to forming a complete PDZ domain. Bridge-1 sequence is less similar to the amino-terminal portions of typical PDZ domains. Bridge- 1 , like ZO- 1 , lacks the conserved sequence GLGF, between beta sheets A and B, that has been identified as the substrate binding site within the third PDZ domain of PSD-95 (Doyle, D. A. et al, Cell 55: 1067-1076 (1996)). Although secondary structure predictions suggest that multiple alpha helices and beta sheets may form within the Bridge- 1 protein, they do not predict a secondary structure pattern that resembles the crystal structures of PDZ domains within PSD-95 or DLG-1 (Cabral, J. H. M, et al, Nature 3352:649-652 (1996); Doyle, O. A. et al, Cell 55:1067-1076 (1996)).

Example 4 Tissue Distribution of Bridge-1 Expression

To study the tissue distribution of Bridge-1 at the RNA level, Bridge-1 cDNA was used as a probe for Northern analysis of RNAs from cells and tissues derived from rodents and humans (FIGs. 3A, 3B, and 3C). Two transcripts of approximately 1.0 kb and 1.3 - 1.4 kb in size are consistently noted in RNA from rodent tissues (FIGs. 3A and 3B), however only a single transcript of approximately 1.0 kb in size is observed in human tissues (FIG. 3C). Bridge-1 RNA is highly expressed in a variety of cell lines derived from pancreatic islets, including the insulinoma line INS-1 (Asfari, M., et al, Endocrinol. 730:167-178 (1992)) from which the cDNA was cloned, and the somatostatin-producing cell line RIN 1027-B2 (Philippe, J.,J. Clin. Invest. 79:351-358 (1987)). Lower levels of expression of Bridge-1 are observed in the glucagon-producing cell lines InRl-G9 and αTCl and the hepatoma cell line HepG2 (FIG. 3 A). Although the expression of Bridge-1 RNA is highest in pancreas, testis, kidney, and liver, expression is detectable in all rodent and human RNA sources tested. Consistent with a widely distributed pattern of Bridge-1 expression are the existence in Genbank databases of several murine and human expressed sequence-tagged cDNA sequences that are homologous to Bridge- 1 and derived from a wide range of tissues, including whole embryos, placenta, brain, central nervous system, heart, and uterus.

To assess the protein expression pattern of Bridge-1, rabbit polyclonal antisera were generated against an internal peptide sequence within Bridge-1. These antisera distinguish the in vitro translated Bridge-1 on Western blots from other proteins present in the rabbit reticulocyte lysate (FIG. 3D). Assessment of

Bridge- 1 expression in RINl 027-B2 cells by immunocytochemistry revealed that Bridge-1 is predominantly located in the nucleus, although lower levels of cytoplasmic staining are observed in some cells (FIG. 4A). Bridge-1 expression did not appear to localize to the cytoplasmic projections extended by the RIN 1027-B2 cells growing in culture. In embryonic day 19 mouse pancreas, nuclear

Bridge-1 immunostaining was prominent within the cells of pancreatic islets, ductal cells, and in a few scattered nuclei of pancreatic exocrine cells (FIG. 4D). A lower level of cytoplasmic Bridge-1 staining was seen in islets but not in the exocrine pancreas. In the adult murine pancreas, Bridge-1 is expressed in pancreatic β-cells, as demonstrated by coexpression with insulin (FIGs. 5 A, 5B).

The observed patterns of Bridge-1 protein expression by immunostaining, with higher levels of endocrine relative to exocrine pancreatic expression and Bridge- 1 expression in pancreatic β-cells, mimic the RNA expression patterns observed in pancreatic cell lines (FIG. 3A). Example 5 Bridge-1 Interaction with E12 and E47

To confirm the observation of the interaction between Bridge-1 and El 2 seen by yeast two-hybrid analysis, additional studies were conducted. Antisera directed against Bridge-1 effectively coimmunoprecipitate in vitro translated

³⁵S-radiolabeled E12 and in vitro translated Bridge-1, as compared with preimmune antisera (FIG. 6A). Both ³⁵S-radiolabeled in vitro translated El 2 and E47 proteins coimmunoprecipitate with in vitro translated Bridge-1. Immunoprecipitations with anti-Bridge-1 antisera conducted in the presence or absence of in vitro translated Bridge- 1 demonstrate the requirement for Bridge- 1 for efficient immunoprecipitation of radiolabeled E12 or E47 (FIG. 6B).

In mammalian two-hybrid studies, fusion protein constructs were transiently transfected into HeLa cells and tested for their ability to activate a GaWCAT reporter (FIG. 7). Fusion protein constructs were generated with the GaWDNA-binding domain or the activation domain of the VP 16 protein from the herpes simplex virus. Empty vectors or the VP16/E12 fusion protein alone have little activity in this system. The GaW DNA-binding domain Bridge- 1 fusion construct has a slight but detectable level of activation of the reporter. However, in the presence of both the NP16/E12 and the GaW DΝA-binding domain/Bridge- 1 fusion constructs, an 18-fold increase in CAT activity is seen.

This activity, in excess of the sum of the activation observed for either fusion construct alone, demonstrates that Bridge-1 interacts with El 2 in living mammalian cells to bring the GaW DΝA-binding domain in proximity to the NP 16 activation domain to activate the transcriptional reporter. Example 6 Bridge-1 Does Not Interact with Beta-2/NeuroD

To determine whether Bridge-1 might be interacting in a non-specific manner with other basic helix-loop-helix proteins, the pancreas-specific transcription factor Beta-2/NeuroD was analyzed in the mammalian two hybrid system. Beta-2 NeuroD is known to directly interact with E2A proteins via heterodimerization of bHLH domains (Mutoh, H., et al, Proc. Natl Acad. Sci. USA 94:3560-3564 (1997); Naya, F. , et al, Genes Dev. 9: 1009-1019 (1995)). As a positive control, Beta-2/NeuroD and El 2 interactions were assessed (FIG. 8). The VP16/E12 and GaW DNA-binding domain Beta-2 fusion proteins together activate the GaWCAT reporter by 15 -fold, a substantially higher activation than that seen for either fusion construct alone. These findings demonstrate and confirm Beta-2/NeuroD interaction with El 2. In contrast, the combination of VP16/B ridge- 1 and GaW DNA-binding domain/Beta-2 fusion constructs did not activate the GaWCAT reporter as compared with either of these two fusion constructs tested individually. Neither mammalian two-hybrid studies nor coimmunoprecipitation studies with in vitro translated proteins demonstrated any interactions between Bridge-1 and Beta-2/NeuroD.

Example 7 Bridge-1 Utilizes its PDZ-like Domain to Interact with E12

To identify the domains within Bridge-1 that mediate its interaction with E2A proteins, Bridge-1 deletion mutants were constructed as GaW DNA-binding domain fusion proteins for analysis of interaction with the VP16/E12 fusion protein construct in mammalian two-hybrid studies. Bridge-1 fusion constructs containing the PDZ domain interacted efficiently with the full length E 12 fusion protein while Bridge-1 constructs without the PDZ-like domain were weak interactors relative to full length Bridge-1 (FIG. 18). Marked overexpression of a mutant Bridge- 1 fusion protein retaining amino acids 1-133 but lacking the PDZ- like domain could produce an interaction with the VP16/E12 fusion construct, raising the possibility that a second weaker cryptic interaction domain may exist within the amino-terminal portion of Bridge-1. In an independent series of experiments by yeast two-hybrid analysis, the original El 2 carboxy-terminal bait (amino acids 521 to 649) was used to assess the strength of interaction of a series of Bridge-1 deletion mutants (FIG. 19). In a pattern analogous to that seen in mammalian cells, Bridge-1 mutants lacking a complete PDZ-like domain failed to efficiently interact with the El 2 bait. In contrast, a mutant lacking the amino-terminal portion of Bridge- 1 but retaining the

PDZ-like domain (amino acids 120-222) demonstrated a potent interaction with El 2. In both mammalian and yeast cells, Bridge-1 requires an intact PDZ-like domain in order to interact with El 2.

Example 8 The Carboxy Terminus of El 2 Contributes to Bridge-1/E12 Interaction

Although the PDZ domain within Bridge-1 has some atypical structural features, it functions as an acceptor site for carboxy-terminal residues of interacting proteins analogous to more typical PDZ-like domains (Saras, J., and C.-H. Heldin, Trends in Biochem. Sci. 27:455-458 (1996)). To test whether the carboxy-terminal amino acids of E 12 contribute to the Bridge- 1 /E 12 interaction, a mutant (E12ΔC) was generated to prematurely truncate El 2 by removing the terminal 9 amino acids but retaining the two activation domains and the basic helix-loop-helix domain (FIG. 10 A). With this mutation, E 12 no longer terminates in a hydrophobic residue. In mammalian two-hybrid analysis, the introduction of this mutation into the VP 16/E 12 fusion construct significantly impairs interaction with the GaW DNA-binding domain/Bridge- 1 fusion construct, decreasing the strength of the E12ΔC interaction with Bridge-1 to 45 percent of that with full length E12 (FIG. 10B). These data indicate that the carboxy terminus of E12 participates in the interaction with Bridge-1.

In contrast, the E12ΔC mutant retains the ability to interact with Beta-2/NeuroD. In mammalian two-hybrid studies, the VP16/E12ΔC fusion construct interaction with the GaW DNA-binding domain/Beta-2 fusion construct is not impaired relative to the VP16/E12 construct interaction with the GaW DNA-binding domain/Beta-2 construct. These results are consistent with Beta-2/NeuroD heterodimerization with E12 via its bHLH domain, a domain left intact in the E12ΔC mutant. Deletion of the bHLH domain and carboxy terminus of El 2, by introduction of a stop codon at amino acid position 527, results in an E12 mutant

(E12ΔbHLH) that is no longer able to interact with Bridge-1 (FIG. 22). This mutant retains both of the activation domains within E12 (FIG. 21). These data indicate that the E12 bait used for yeast two-hybrid screening (amino acids 521 - 649) encompasses all of the E12 domains that participate in the interaction of El 2 and Bridge-1.

Example 9 Bridge-1 Has Intrinsic Transactivation Potential

A small but detectable level of activity was observed for the GaW DNA-binding domain/Bridge- 1 fusion construct alone in transient transfections in

HeLa cells (FIG. 7), supporting the idea that Bridge-1 might have intrinsic transactivation activity independent of interactions with E2A. The activity of this construct is considerably higher in transient transfections of BHK cells (FIG. 11 ), indicating that Bridge-1 activation is a regulated function that varies with differences in intracellular signaling. In BHK cells, the GaW DNA-binding domain/Bridge- 1 fusion construct activates the GaWCAT reporter 28 -fold, as compared to activation of the reporter by the empty vector containing the GaW DNA-binding domain alone. Example 10

Bridge-1 Coactivates Insulin Promoter Elements with E12 and E47

To test the activity of Bridge-1 on mammalian promoter regulatory elements, we utilized a CAT reporter regulated by upstream multimerized E and A box (FarFlat) minienhancers of the rat Insulin I gene (5FF1 CAT). In transient transfections of HeLa cells with equal amounts of expression vectors for Bridge- 1 and E47, activation of the 5FF1CAT reporter was assessed (FIG. 12A). Transfection of E47 alone minimally activates the reporter 1.4-fold, and transfection of Bridge-1 alone results in a 1.5-fold activation. However, transfection of the two constructs in combination results in a 5.2-fold activation of the reporter, demonstrating that Bridge-1 enhances E47 transactivation. Transfection of increasing amounts of Bridge-1 expression vector also increases E12-mediated activation of the FarFlat reporter in a dose-dependent manner up to 24-fold (FIG. 12B). The E12ΔC mutation that impairs Bridge-1/E12 interaction decreases, by 65%>, the combined activity of Bridge-1 and E12 on the

FarFlat reporter (FIG.23). The strength of Bridge- 1 interaction with E 12 appears to modulate the level of the observed coactivation. Western blotting of extracts from these transfected cells demonstrates that the differences in coactivation are not a result of significant differences in expression of E 12 and E 12ΔC (FIG. 12D). No significant difference was observed in the activation of 5FF 1 CAT by E 12ΔC versus E12 in transfections conducted without the addition of Bridge-1.

In selected DNA-binding assays using a FarFlat oligonucleotide probe, no direct binding of either in vitro translated or recombinant Bridge-1 was observed. In similar studies, anti-Bridge- 1 antisera did not attenuate DNA-binding of protein complexes from insulinoma cell nuclear extracts, although attenuation of protein-binding was observed with anti-E2A antiserum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Endogenous Bridge-1 inactivation impairs insulin promoter activity in INS-1 cells. To determine whether endogenous Bridge-1 levels are important in the regulation of insulin promoter activity, an antisense Bridge- 1 cDN A construct, AS-Bridge-l-pcDNA3, was employed in transient transfections of the INS-1 rat insulinoma cell line. A promoter-reporter construct consisting of -410 to +47 rat insulin I promoter sequences, -410INS-LUC (Lu, M. et al., J. Biol. Chem. 272:28349-28359 (1997)), was utilized to assess the effect of expression of the antisense Bridge-1 construct. Expression of the AS-Bridge-l-pcDNA3 construct decreased insulin promoter activity by 45%) (FIG.24). These results indicate that endogenous Bridge-1 contributes to insulin promoter activation in insulin- producing cells.

Example 11

Bridge-1 signaling is operative during the development of the pancreas.

Bridge- 1 expression during early pancreas development was demonstrated by three independent methods. First, the complete Bridge-1 cDNA was isolated by screening a rat 14 dpc pancreatic library, indicating that Bridge-1 is expressed at this stage of pancreatic development in the rat. Second, by rt-PCR, we amplified a 403 nucleotide Bridge-1 fragment (nucleotides 553-955) from cDNA prepared from microdissected pooled mouse pancreas samples at embryonic days 10.5 and 13.5, indicating that Bridge- 1 is expressed in developing mouse pancreas at these stages. In these experiments, Bridge-1 expression at el 0.5 was detected in a cDNA sample from which PDX-1 expression was too low to detect; at el 3.5 both PDX-1 and Bridge-1 expression were observed. Bridge-1 expression appears to be more marked than PDX-1 expression at a very early stage of pancreas development. Third, polyclonal rabbit anti-Bridge-1 antisera (directed against an internal peptide epitope within Bridge-1 ) was used in immunostaining of paraffin sections of developing mouse pancreas. At embryonic day 15, Bridge-

1 is expressed in a nuclear pattern throughout the branching, developing ductal tree destined to become pancreas, as well as within structures budding from the ducts that likely represent developing islets. At this stage of pancreas development, the Bridge-1 expression pattern closely mimics that of PDX-1. Of note, the homeodomain transcription factor PDX-1 is implicated in the pathogenesis of forms of pancreatic agenesis and diabetes mellitus in mice and humans.

Example 12

Bridge-1 interacts with PDX-1

In the original cloning of Bridge- 1 in the yeast two-hybrid system, Bridge- 1 was tested with several other fusion protein constructs for interaction. Bridge- 1 interacted with a fragment of E12 and did not interact with the protein bicoid or the interleukin-1 receptor in this system, as indicated in the first manuscript.

However, when Bridge-1 was tested for interaction with a fragment of PDX-1 (amino acids 160-283), a strong interaction was observed, both by growth on leucine dropout plates and by the growth of intense blue colonies on X-gal plates (FIG. 14a). This interaction was verified in the mammalian two hybrid system, although the interaction is weaker in this system than the Bridge- 1/E12 interaction

(FIG. 14b). The functional consequences of the Bridge-1 /PDX-1 interaction are currently under investigation, but Bridge- 1 modulation of PDX- 1 function is likely to be directly relevant to PDX-1 regulation of pancreas development, PDX-1 regulation of insulin gene transcription (known to occur in synergy with El 2, an additional target of Bridge- 1 coactivation and interaction), and PDX- 1 regulation of differentiated pancreatic beta cell function.

Example 13 Bridge-1 transactivation utilizes its PDZ-like domain and carboxy-terminal sequences

Mutants of the Bridge-1 protein fused to the GaW DNA binding domain were tested for the ability to transactivate a GaWCAT reporter. In this manner, map domains of Bridge-1 required for transactivation activity were possible. In these studies, deletion of the carboxylterminus of Bridge- 1 results in severe impairment of transactivation activity, while preserving Bridge- 1/E12 interaction activity. Deletion of amino acids 186-222 or even of amino acids 215-222 impairs Bridge- 1 transactivation. Larger carboxy-terminal deletion mutants that lack the

PDZ-like domain (retaining amino acids 1-73, for example), lack Bridge-1 transactivation activity. Interestingly, a series of point mutants were made within the 41 amino acid domain of Bridge-1 identified as the PDZ-like domain. These mutants were designed to disrupt conserved residues within the PDZ-like domain. Seven distinct point mutations of conserved residues within the PDZ-like domain disrupt Bridge-1 transactivation (five examples are shown in FIG. 15). (Many of these mutants also disrupt Bridge-1 interaction with El 2 in the mammalian two- hybrid assay, confirming the data in the manuscript that indicates that the Bridge- 1 PDZ-like domain is a mediator of protein-protein interactions.) These data indicate that integrity of the PDZ domain is required for both Bridge-1 transactivation activity and interaction with El 2.

Some differences in relative impairment of interaction versus transactivation activities have been observed in mutagenesis studies, indicating that these two activities will have somewhat different requirements. For example, the Bridge- 1 (1-185) mutant retains the ability to interact with E 12 but has very poor transactivation activity.

The utilization of the PDZ-like domain for Bridge- 1 transactivation activity indicates that targeting inhibition of this domain with small molecule inhibitors will alter Bridge-1 activity. Further, the observation that different structural requirements specify transactivation versus interaction activities indicates that it is possible to design small molecule inhibitors that specifically target distinct activities of the Bridge- 1 protein. Since Bridge- 1 is a key regulator of insulin gene transcription, such inhibitors should modulate insulin gene transcription and be of therapeutic relevance to the disease diabetes mellitus.

Example 14 Bridge-1 function is modulated by p300

The ability of Bridge-1 to interact with coactivators with histone acetyl transferase activity that interact with basal transcriptional machinery, such as CBP and p300, was tested. The Bridge-1/GaW DNA binding domain fusion protein construct transactivting a GaWCAT reporter was utilized as our test system in transient transfections in which we added increasing amounts of p300. In a dose- dependent manner, the addition of p300 increased Bridge-1 transactivation activity, as much as 25-fold (FIG. 16). These data indicate that Bridge-1 and p300 work together to transactivate transcriptional targets.

In additional preliminary experiments on the rat Insulin I promoter-reporter construct FarFlat-C AT, coactivation of Bridge- 1 of E 12-mediated transcriptional activation was repressed by cotransfection of CBP or p300. These data indicate that Bridge-1 transactivation or coactivation is modulated by interactions with additional coactivators.

Example 15 Multiple forms of Bridge-1 exist.

A number of observations indicate that alternate forms of Bridge- 1 are operative in Bridge-1 signaling. In tissues derived from mice and rats, two

Bridge- 1 mRNAs of different sizes are routinely observed, indicating that multiple Bridge-1 transcripts are made, possibly resulting in different forms of Bridge-1 protein. In Genbank databases, human homologues of Bridge-1 have different carboxy-terminal sequences, indicating that alternate splicing of the carboxyl terminus occurs. Of note, this splice junction occurs out of frame and hence entirely changes the carboxy-terminal Bridge- 1 amino acid sequence . This splicing occurs at a position near the site of the carboxy-terminal deletion mutation (1-186) that disrupts Bridge-1 transactivation activity. Since we have implicated the carboxyl terminus of Bridge- 1 as essential for transactivation activity, changing its amino acid sequence by alternate splicing mechanisms will affect transactivation activity. The implication of this finding is that small molecules that target distinct forms of carboxy-terminal Bridge-1 sequences have different biological effects.

In addition to the evidence for different Bridge-1 transcripts, multiple forms of Bridge-1 were detected with rabbit polyclonal antisera on Western blots of cellular extracts. The full length in vitro translated Bridge-1 protein migrates at approximately 28-29 kilodaltons on Western blots. On Western blots of cellular extracts, a 28-29 kilodalton protein is detected, but often a protein of approximately 18 kilodaltons in size is also detected (FIG. 17). The ratio of this "small form" of Bridge-1 relative to the full length protein is high for multiple insulinoma cell lines, intermediate for the hamster fibroblast cell line BHK, and low for the human cell line HeLa. Although it is unclear whether the " small form" of Bridge-1 represents an alternate splice form or a smaller Bridge-1 protein derived from proteolysis of the full length Bridge-1, the expression pattern observed for "small form" suggests that it may be important in mediating Bridge- 1 actions in pancreatic beta cells. One observation suggesting that the "small form" of Bridge-1 may result from proteolysis was that combination of radiolabeled full length in vitro translated Bridge-1 with a GST-PDX- 1 preparation in GST pull-down assays resulted in GST-PDX- 1 association with a radiolabeled protein of approximately 18 kilodaltons reminiscent of the "small form" of Bridge-1 observed in Western blots. These data suggest that under some circumstances full length Bridge-1 protein can be converted to "small formJ

Discussion

A novel protein that interacts with El 2 by yeast two-hybrid analysis has been identified. The protein is designated Bridge-1 to reflect its probable role as a coactivator that functions via protein-protein interactions. Consistent with this concept of protein-protein interactions is the localization within Bridge-1 of a truncated PDZ-like domain that is required for its interaction with E12. Whereas most characterized PDZ domain-containing proteins have been localized to membrane or cytoplasmic compartments, a small number of nuclear proteins with

PDZ domains have been identified. The PDZ-like domain within Bridge-1 may be a subtype that functions within the nucleus, as it is similar to PDZ domains within the nuclear protein SIP- 1. SIP- 1 interacts with the testis determining factor SRY and has an identical sequence to proteins designated TKA- 1 (tyrosine kinase activator- 1, Genbank ace. no. Z50150) and E3KARP (NH3 kinase A regulatory protein (Zhuang, Y., et al, Cell 79:875-884 (1994))). Other proteins with PDZ domains homologous to Bridge- 1 include a Tax-binding protein (Zhuang, Y., et al, Cell 79:875-884 (1994)), and human proteins of unknown function with leucine zipper or LIM domain motifs usually found in transcription factors (FIG. 2C). The PDZ-like domain within Bridge-1 also shares homology with cytoplasmic proteins, including a regulator of renal Na+/H+ exchange, proteases of the DEGS type and the tight junction protein ZO-1.

The PDZ-like domain of Bridge-1 has some atypical structural features. The stretch of PDZ homology within Bridge-1 is 41 amino acids, approximately half the size of typical PDZ domains (Cabral, J. H. M., et al, Nature 3352:649-

652 (1996); Doyle, D. A. et al, Cell 55:1067-1076 (1996)). It is possible that additional regions within Bridge- 1 may contribute secondary structure to complete the PDZ domain. The absence of conservation within Bridge-1 of the peptide binding site described in the third PDZ domain of PSD-95 (Doyle, D. A. et al, Cell 55:1067-1076 (1996)) is of interest. However, alterations in substrate binding site configurations within PDZ domains are likely needed to provide specificity for protein-protein interactions. Bridge-1 may contain a distinct type of PDZ-like domain with different structural determinants.

The identification, by yeast two-hybrid screening using the same E12 carboxy terminal bait, of a second novel PDZ domain-containing protein PIN- 1 (clone # 36) (Yao and Wong, unpublished results), indicates that Bridge- 1 is apart of a larger signaling network involving E2A/PDZ domain communication. Future studies of Bridge-1 and PIN-1 function should provide opportunities to define signaling pathways that may regulate E2A function.

E12 and E47 interact with other basic helix-loop-helix proteins through heterodimerization via helix-loop-helix domains (Murre, C, et al, Biochim. Biophys. Acta 7275:129-135 (1994)). The yeast two-hybrid bait used to clone Bridge- 1 was derived from carboxy terminal E 12 sequences, including the bHLH domain and the carboxy terminus (FIG. 10A). Although the Bridge-1 protein sequence includes regions of hydrophobicity, homologies with basic helix-loop-helix proteins were not observed. In addition, the failure of Bridge-1 to interact with Beta-2, under experimental conditions in which Bridge- 1/E12 and Beta-2/E12 interaction occur, indicates that Bridge- 1/E2A protein interactions are highly specific. Deletion of 9 carboxy terminal amino acids from El 2, in the mutant E12ΔC, substantially diminished Bridge-1 and E12 interactions but did not interfere with the interaction of Beta-2/NeuroD with E12. In general, PDZ domains interact with carboxy-terminal sequences within target proteins (Saras, J., and C.-H. Heldin, Trends in Biochem. Sci. 27:455-458 (1996)). Truncation of the El 2 carboxy terminus markedly reduced its interaction with Bridge-1, indicating that the model of PDZ domain interaction with carboxy terminal sequences also applies to the Bridge-1 /El 2 interaction. Although it is possible that other regions within El 2 stabilize its interaction with Bridge-1, the E12 carboxy terminus is an important mediator of this protein-protein interaction, supporting a Bridge- 1 /El 2 interaction model distinct from the bHLH heterodimerization model utilized by most E12 interacting proteins.

The El 2 carboxy terminus is important both for interaction with Bridge-1 and for Bridge- 1 coactivation of El 2. Bridge-1 coactivation of El 2-mediated transactivation of the insulin promoter enhancer sequence FarFlat was impaired by approximately 65%o in the absence of the nine carboxy-terminal amino acids of El 2. These data support a model in which interaction of the carboxy-terminal domain of El 2 with the PDZ-like domain of Bridge-1 results in increased transcriptional activation of El 2 targets. This Bridge-1 -El 2 interaction model is distinct from typical heterodimerization models thought to be employed by most

El 2 interacting proteins.

Other models of protein-protein interaction for E2A proteins, that do not involve heterodimerization via helix-loop-helix domains, are supported by reports of E2A interactions with the polymyositis-scleroderma autoantigen and the ubiquitin-conjugating enzyme, UbcE2A/mUBC9 (Kho, C.-J.. etal, J. Biol. Chem.

272:3845-3851 (1997); Kho, C.-J., et al, J. Biol Chem. 272:13426-13431 (1997); Loveys, D. A., etal, Gene 207:169-177 (1997)). These proteins interact with internal E12 sequences distinct from the carboxy terminus. The interaction of E2A proteins with UBcE2A/mUBC9 is intriguing, in light of the designation, human proteasomal modulator subunit p27, attributed to the human homologue of Bridge-1 (Watanabe, T. K, et al, Genomics 50:241-250 (1998)). Additional evidence for the potential involvement of components of a regulated protein degradation pathway in E2A signaling was provided by a recent report that the proteasomal subunit S5a regulates the binding of E12 and MyoD to DNA by direct interactions with Idl (Anand, G., X. et al, J. Biol Chem. 272: 19140-

19151 (1997)). Further studies are required to determine whether Bridge-1 may function both as an E2A coactivator and in some additional capacity as a regulator of E2A degradation.

The original intent in screening an insulinoma cell cDNA library was to identify factors within the endocrine pancreas that may modulate E2 A function on β-cell genes. However, the wide distribution of Bridge-1 expression resembles that of the E2A proteins and indicates that it plays a broader role in the modulation of E2A activity. Furthermore, the level of protein conservation of Bridge- 1 across species implies a fundamental function of biological importance. It is tempting to speculate that Bridge-1 functions in developing tissues, in light of the embryonic expression of cDNAs homologous to Bridge-1 reported in databases of expressed sequence tags derived from embryonic tissues and Bridge- 1 expression in the 14 dpc rat pancreatic cDNA library. Because E2A null mice have abnormalities of lymphocyte development (Bain, G., etal, Cell 79:885- 892 (1994); Mutoh, H., et al, Proc. Natl. Acad. Sci. USA 94:3560-3564 (1997)), and mice nullizygous for beta-2/neuroD, the pancreas-specific dimerization partner of E2A, demonstrate abnormalities in pancreas development (Naya, F. J., et al, Genes Development 11 :2323-2334 (1997)), proteins that modulate the transactivation functions of E2A are candidate developmental regulators. Bridge-1 functions as a strong activator in the context of its fusion to the

GaW DNA-binding domain. The difference in the activity of this construct in the two cell types tested indicates that this transactivational activity is a regulated function. Possible explanations for these differences in activity include different post-translational modifications of Bridge- 1 protein that alter its conformation and function, or differences in expression patterns of protein-binding partners for

Bridge-1. The transactivation data are consistent with either an intrinsic transactivation domain within Bridge-1 or a recruitment function that attracts additional transactivating proteins.

Bridge-1 is important in the pancreas in regulating the insulin and other islet genes. A high level of Bridge-1 expression in pancreatic islet cell lines was observed compared to a cell line derived from exocrine pancreas, as well as expression predominantly in the islets in sections of mouse pancreas. Of note, Bridge-1 is expressed in the insulin-producing β-cells of murine islets. The nuclear localization of Bridge-1 is consistent with its function as a coactivator. E2A regulates the promoter activities of islet-specific genes, including the insulin and glucagon genes, via binding to E boxes within the promoters of these genes (Cordier-Bussat, M., etα/., Mol. Cell. Biol. 75:3904-3916 (1995)). In the insulin gene, E boxes in the promoter are partially responsible for glucose-responsive transcription (German, M. S., and J. Wang, Mol. Cell. Biol. 74:4067-4075 (1994)). Previous investigators have demonstrated that E2A proteins heterodimerize with the tissue-specific bHLH transcription factor Beta-2/NeuroD in binding to the E boxes of the insulin promoter (Naya, F. J., et al, Genes Dev. 9: 1009-1019 (1995)). These heterodimers work in synergy with homeodomain proteins, including PDX- 1 , to activate the insulin promoter via minienhancers such as FarFlat (German, M. S. et al, Genes Dev. 6:2X65-2X16 (1992); Peers, B., et al, Mol. Endo. 5:1798-1806 (1994)). Bridge-1 enhanced the activation of this minienhancer by either E 12 or E47. The lack of DNA-binding activity of Bridge- 1 on FarFlat oligonucleotides is consistent with a role for Bridge-1 in this context as a coactivator rather than a direct transactivator. The absence of any obvious DNA-binding domain within the Bridge-1 sequence supports this model. It is possible that, with other target DNA sequences, or under other conditions, Bridge-1 might have a cryptic DNA-binding function.

The Bridge-1 coactivation of E12-mediated FarFlat transactivation is largely dependent on an intact carboxy terminus within El 2, indicating that Bridge-1 /El 2 interaction mediates the observed coactivation. The coactivator p300 is proposed to stimulate insulin gene transcription via direct interactions with E47 and Beta-2/NeuroD (Qiu, Y., et al, Mol. Cell Biol 75:2957-2964 (1998)). The present data show that Bridge-1 coactivation of E12 is occurs through a mechanism independent of Beta-2/NeuroD. The physiologic relevance of the regulation of the glucose-responsive regions of the insulin promoter is underscored by the recent reports that inactivating mutations in proteins that regulate this response, such as PDX-1 , are linked to diabetes mellitus in humans (Staffers, D. A., et al, Nature Genet. 77: 138-139 (1997)). The observation that Bridge-1 acts with E12 and E47 to increase the activation of a critical enhancer in the insulin promoter provides an alternative model for E2A activation and may permit the elucidation of novel mechanisms of insulin gene regulation.

Example 16

Therapeutic Aspects of the Invention

In one therapeutic aspect of the invention polypeptides, biological or chemical compounds or other small molecules that enhance or inhibit Bridge- 1 activity are used to treat diabetes or other developmental disorders by modifying the transcription activation of target genes including the insulin gene.

In another therapeutic aspect of the invention, an animal may be treated by introducing into the animal one or more of the isolated nucleic acid molecules of the invention comprising a polynucleotide encoding a mammalian Bridge-1 polypeptide or a fragment thereof, particularly a polynucleotide that is 90% or 95%) identical to the Bridge-1 nucleotide sequence or a Bridge-1 fragment nucleotide sequence, such as a fragment encoding the PDZ-like domain and/or the carboxyl terminus domain of Bridge-1. This approach, known generically as

"gene therapy ' is designed to increase the level of mammalian Bridge-1 gene expression in the cells affected by or causing the disorder (such as cancer or tumor cells) and thereby to cure, delay or prevent the development of, or induce remission of, the disorder by restoring cell cycle checkpoint control to the affected cells. Analogous gene therapy approaches have proven effective or to have promise in the treatment of certain mammalian diseases such as cystic fibrosis (Drumm, M.L. et al, Cell (52:1227-1233 (1990); Gregory, R.J. et al, Nature 347:358-363 (1990); Rich, D.P. et al, Nature 347:358-363 (1990)), Gaucher's disease (Sorge, J. etal, Proc. Natl. Acad. Sci. USA 54:906-909 (1987); Fink, J.K. et al, Proc. Natl. Acad. Sci. USA 57:2334-2338 (1990)), certain forms of hemophilia (Bontempo, F.A. et al, Blood (59:1721-1724 (1987); Palmer, T.D. et al, Blood 73:438-445 (1989); Axelrod, J.H. et al, Proc. Natl. Acad. Sci. USA 57:5173-5177 ( 1990); Armentano, D. et al. , Proc. Natl. Acad. Sci. USA 87:6X4X- 6145 (1990)) and muscular dystrophy (Partridge, T. A. et al. , Nature 337: X 76- 179 (1989); Law, P.K. et al, Lancet 336: 114-115 (1990); Morgan, ].E. et al, J. Cell Biol. 777:2437-2449 (1990)), as well as in other treatments for certain cancers such as metastatic melanoma (Rosenberg, SA. et al, Science 233: 1318-1321 (1986); Rosenberg, S.A. et al, N. Eng. J. Med. 379:1676-1680 (1988);

Rosenberg, S.A. et al, N. Eng. J. Med. 323:570-578 (1990)).

In a preferred such approach, one or more isolated nucleic acid molecules of the invention, comprising a polynucleotide having a nucleotide sequence at least 90% or at least 95%) identical to the Bridge-1 sequence or a Bridge-1 fragment, such as a fragment encoding the PDZ-like domain, is introduced into or administered to the animal that is suffering from or predisposed to the disorder. Such isolated nucleic acid molecules may be incorporated into a vector or virion suitable for introducing the nucleic acid molecules into the cells or tissues of the animal to be treated, to form a transfection vector. Suitable vectors or virions for this purpose include those derived from retroviruses, adenoviruses and adeno- associated viruses. Alternatively, the nucleic acid molecules of the invention may be complexed into a molecular conjugate with a virus (e.g., an adenovirus or an adeno-associated virus) or with viral components (e.g., viral capsid proteins).

Techniques for the formation of vectors or virions comprising the mammalian Bridge- 1 -encoding nucleic acid molecules are well-known in the art, and are generally described in "Working Toward Human Gene Therapy," Chapter 28 in Recombinant DNA, 2nd Ed., Watson, J.D. etal, eds., New York: Scientific American Books, pp. 567-581 (1992). In addition, general methods for construction of gene therapy vectors and the introduction thereof into affected animals for therapeutic purposes may be obtained in the above-referenced publications, the disclosures of which are specifically incorporated herein by reference in their entirety. In one such general method, vectors comprising the isolated polynucleotides of the present invention are directly introduced into the cells or tissues of the affected animal, preferably by injection, inhalation, ingestion or introduction into a mucous membrane via solution; such an approach is generally referred to as "in vivo" gene therapy. Alternatively, cells, tissues or organs, particularly those containing cancer cells or tumors, may be removed from the affected animal and placed into culture according to methods that are well- known to one of ordinary skill in the art; the vectors comprising the mammalian Bridge- 1 polynucleotides may then be introduced into these cells or tissues by any of the methods described generally above for introducing isolated polynucleotides into a cell or tissue, including viral infection or transfection, and, after a sufficient amount of time to allow incorporation of the Bridge- 1 polynucleotides, the cells or tissues may then be re-inserted into the affected animal. Since the introduction of the mammalian Bridge- 1 gene is performed outside of the body of the affected animal, this approach is generally referred to as "ex vivo" gene therapy.

For both in vivo and ex vivo gene therapy, the isolated mammalian Bridge- 1 polynucleotides of the invention may alternatively be operatively linked to a regulatory DNA sequence, which may be a mammalian Bridge-1 promoter or an enhancer, or a heterologous regulatory DNA sequence such as a promoter or enhancer derived from a different gene, cell or organism, to form a genetic construct as described above. This genetic construct may then be inserted into a vector, which is then directly introduced into the affected animal in an in vivo gene therapy approach, e.g., by intratumoral administration (i.e., introduction of the nucleic acid molecule or vector directly into a tumor in an animal, for example by injection), or into the cells or tissues of the affected animal in an ex vivo approach. In another preferred embodiment, the genetic construct of the invention may be introduced into the cells or tissues of the animal, either in vivo or ex vivo, in a molecular conjugate with a virus (e.g., an adenovirus or an adeno-associated virus) or viral components (e.g. , viral capsid proteins; see WO 93/07283). These approaches result in increased production of Bridge-1 polypeptide by the cells of the treated animal via (a) random insertion of the Bridge-1 gene into the host cell genome; or (b) incorporation of the Bridge-1 gene into the nucleus of the cells where it may exist as an extrachromosomal genetic element. General descriptions of such methods and approaches to gene therapy may be found, for example, in U.S. Patent No. 5,578,461 ; WO 94/12650; and WO 93/09222.

Regardless of the approach used, however, use of these methods of the present invention will result in the increased production of Bridge-1 polypeptide by the cells and tissues of the treated animal, such that the disorder will be delayed or inhibited, or such that the disorder will go into remission or be cured.

As an alternative to the above, one may administer the isolated or recombinant Bridge-1 protein or Bridge-1 fragment to the animal to increase or decrease the level of endogenous insulin in the cells and tissues of the animal as needed.

These methods may be used to treat disorders resulting from overexpression or underexpression of Bridge-1, including diabetes.

All patents and publications referred to above are hereby entirely and expressly incorporated herein by reference. Narious modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the inventions as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the pertinent art are intended to be within the scope of the claims.

Claims

What Is Claimed Is:

X . An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95%> identical to a sequence selected from the group consisting of: (a) a polynucleotide fragment of the coding region of SEQ ID

NO: 1;

(b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO: 2;

(c) a polynucleotide encoding a polypeptide domain of SEQ ID NO: 2;

(d) a polynucleotide encoding a polypeptide epitope of SEQ ID NO: 2;

(e) a polynucleotide encoding a polypeptide of SEQ ID NO: 2 having biological activity; (f) a polynucleotide which is a variant of the coding region of

SEQ ID NOT ; (g) a polynucleotide which is an allelic variant of the coding region of SEQ ID NOT; (h) a polynucleotide which encodes a species homologue of SEQ ID NO:2;

(i) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h), wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.

2. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a mature form or a secreted Bridge-1 protein.

3. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO:2.

4. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises the entire open reading frame of the nucleotide sequence of SEQ ID NO: 1.

5. The isolated nucleic acid molecule of claim 2, wherein the nucleotide sequence comprises sequential nucleotide deletions from either or both the C-terminus or the N-terminus.

6. The isolated nucleic acid molecule of claim 3, wherein the nucleotide sequence comprises sequential nucleotide deletions from either or both the C-terminus or the N-terminus.

7. An isolated polynucleotide which comprises a nucleotide sequence that is complementary to a polynucleotide fragment of claim 1.

8. A method for making a recombinant vector comprising inserting the isolated nucleic acid molecule of claim 1 into a vector.

9. A recombinant vector comprising the isolated nucleic acid molecule of claim 1.

10. A method of making a recombinant host cell comprising introducing the recombinant vector of claim 9 into a host cell.

11. A host cell comprising the vector of claim 9.

12. An isolated polynucleotide which encodes a fragment of at least 40 amino acids of the amino acid sequence in SEQ ID NO: 2.

13. An isolated polynucleotide according to claim 12 which comprises the open reading frame shown in SEQ ID NO: 1.

14. The isolated polynucleotide of claim 12 which comprises the nucleotide sequence encoding amino acids 138-178 in SEQ ID NO: 2.

15. A vector which comprises a polynucleotide according to claim 12 or claim 13.

16. A host cell which comprises a vector according to claim 15.

17. The isolated nucleic acid molecule of claim 1 wherein said polynucleotide is DNA.

18. The isolated polynucleotide of claim 17 wherein said polynucleotide is cDNA.

19. A vector which comprises an isolated nucleic acid molecule according to claim 13.

20. A host cell comprising a vector according to claim 19.

21. An expression vector which comprises a polynucleotide having an open reading frame operably linked to a promoter, said open reading frame encoding a polypeptide selected from the group consisting of: (a) a polypeptide having the amino acid sequence set forth in

SEQ ID NO: 2; (b) a polypeptide having the amino acid sequence corresponding to amino acids 138-178 in SEQ ID NO: 2;

(c) an allelic variant or mammalian homologue of the amino acid sequence set forth in SEQ ID NO: 2; (d) a polypeptide having at least 80%> homology to the amino acid sequence set forth in SEQ ID NO: 2;

22. A host cell transformed by the expression vector of claim 21.

23. A method for preparing a polypeptide encoded by the expression vector of claim 21 , said method comprising cultivating a host cell transformed by said expression vector under conditions that provide for expression by said vector, and recovering the expressed polypeptide.

24. A recombinant polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.

25. A recombinant polypeptide comprising amino acids 138- 178 of the amino acid sequence shown in SEQ ID NO: 2.

26. An isolated nucleic acid probe encoding at least 40 contiguous amino acids of the amino acid sequence in SEQ ID NO: 2.

27. A method for identifying or isolating a protein, protein fragment, biological or chemical compound or other small molecule that binds to Bridge- 1 or a Bridge-1 fragment comprising contacting a solution containing Bridge-1 or Bridge-1 fragment with at least one protein, protein fragment, biological or chemical compound or other small molecule of interest.

28. A method of stimulating gene expression in a cell comprising transfecting a cell with the recombinant vector of claim 9.

29. A method of stimulating insulin gene expression in a cell comprising transfecting a cell with the recombinant vector of claim 5.

30. A method of treating diabetes comprising administering to a patient in need thereof the recombinant polypeptide of claim 24 or claim 25 in a pharmaceutically acceptable carrier or excipient for an amount of time sufficient to provide an effective level of endogenous insulin in the patient.

31. An isolated antibody that binds specifically to the recombinant polypeptide according to claim 24 or 25.

32. A nucleic acid molecule that hybridizes to the isolated polynucleotide of claim 1 under stringent conditions, wherein said stringent conditions are overnight incubation at 42 °C in a solution comprising 50% formamide, 5xSSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10%> dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1xSSC at about 65°C.

33. The isolated polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid is replaced by a conservative amino acid substitution.

34. A method of treating diabetes comprising:

(a) transfecting a cell with the recombinant vector of claim 9; and

(b) stimulating insulin expression in said cell.

35. A method for stimulating expression of a Bridge- 1 target gene by administering an effective amount Bridge-1.

36. A method for enhancing or inhibiting expression of a Bridge-1 target gene by administering an effective amount of a protein, protein fragment, biological or chemical compound or other small molecule that enhances or inhibits Bridge-1 activity.